In sexually reproducing organisms a new individual is formed when two haploid cells, the spermatozoon and the egg, come together to form a diploid zygote. In most internally fertilizing species, the spermatozoa are deposited in the lower regions of the female reproductive tract and, from there, migrate up to the higher reaches of the tract where sperm–egg fusion and zygote formation result. Before fusion and zygote formation can occur, the spermatozoon must bind to, and penetrate, an extracellular coat that surrounds the egg. Once binding and penetration have taken place, the spermatozoon can fuse with the cell membrane of the egg, the oolemma, resulting in syngamy and oocyte activation. An acellular coat around recently ovulated eggs is present in all vertebrates and even around some inverte- brate eggs, such as those of sea urchins. In vertebrates, this coat is variously referred to as the chorion in fish, the vitelline envelope in amphibians, the perivitelline envelope in reptiles and birds, and the zona pellucida in both eutherian and marsupial mammals. Although the general morphology of the coat is similar in all of these vertebrate lineages, its thickness varies greatly among the major groups. In marsupials, the zona pellucida tends to be somewhat thinner than in eutherians (Fig. 1) and this feature may be associated with a more lytic process of zona pene- tration than that which occurs in eutherian mammals (Bedford, 1991, 1998). In this review, the recently acquired knowledge of the structure and function of the zona pellucida of marsupials is compared with that already documented in eutherian mammals. Although several functions of the eutherian zona pellucida are fairly well established, the organization of the structural components, and their evolutionary relationships, remain equivocal. It has generally been believed that the eutherian zona pellucida is composed of three sulphated glycoproteins, ZPA (ZP2 in rodents), ZPB (ZP1 in rodents), and ZPC (ZP3 in rodents) (for example, Wassarman, 1988; Dunbar et al., 1994; Epifano et al., 1995, Wassarman et al., 1999). (The terms ZPA, ZPB and ZPC are used in the present review to describe the major glycoprotein components of the zona pellucida as proposed by Harris et al., 1994.) It has been assumed that comparisons between, for example, human and mouse ZPB are comparisons between orthologous genes, that is, genes that trace their origin to a single gene present in the most recent common ancestor. Recent work on laboratory mice and humans indicates that such a view may be oversimplistic and that a gene, named ZP1 to distinguish it from human ZPB, is the true human orthologue of the mouse ZPB (ZP1) gene (Hughes and Barratt, 1999). In the context of these uncertainties and the Structural organization and evolution of the marsupial zona pellucida William G. Breed 1 , Rory M. Hope 2 , Ole W. Wiebkin 3 , Scott C. Spargo 2 and Jamie A. Chapman 1 1 Department of Anatomical Sciences, 2 Laboratory of Molecular Evolution, Department of Molecular Biosciences and 3 Department of Medicine, The University of Adelaide, Adelaide, South Australia 5005, Australia In this review, the biochemical composition and structural organization of the marsupial and eutherian zonae pellucidae are compared. Differences between the zonae from these two groups of mammals are observed in their response to dilute proteases and reducing agents, in their potential glycosylation patterns, and in some of their functions. However, studies on the glycoconjugates and polypeptides of the three zona pellucida genes have not explained these different responses to the proteases and reducing agents. There is high sequence similarity between the zona polypeptides of marsupials and eutherians, as well as a similarity in the oligosaccharides present, as demonstrated by lectin staining. As the marsupial and eutherian lineages diverged from a common ancestor over 100 million years ago, these observations indicate that the three-dimensional structure of these glycoproteins is highly conserved throughout all mammals, although the complexity of its molecular organization has yet to be resolved. Phylogenetic analyses indicate that there are at least four groups of paralogous zona pellucida genes in vertebrates. The marsupial ZPA and ZPB genes have been named in accordance with their orthologues but the phylogenetic relationships of the marsupial ZPC gene require further investigation. © 2002 Society for Reproduction and Fertility 1470-1626/2002 Reproduction (2002) 123, 13–21 Review Email: bill.breed@adelaide.edu.au