1. Introduction During the last decade, the improvement of compar- ative cytogenetics has been closely linked to tech- nical and methodological advances in fluorescence in situ hybridization (FISH) technology [1–4]. These advances combine chromosome sorting by flow cytometry with subsequent DNA amplification and labeling of sorted chromosome probes by DOP-PCR (degenerate oligonucleotide-primed PCR) or PARM- PCR (priming authorizing random mismatches PCR) using primers that are not species-specific [5, 6]. Both PCR methods allow the production of chromo- some-specific painting probes from various species, which were subsequently used for homologous and heterologous chromosome painting thus permitting the comparison of several karyotypes between closely and distantly related species [7–12]. Rat, which belongs to the Muridae, is widely used in biomedical and pharmaceutical research but up to now, its genome was significantly less studied than the mouse genome. As Muridae species under- went an intense chromosome evolution with the occurrence of many translocations and complex structural rearrangements, it is not possible to directly transpose the mouse genome knowledge to that of the rat [13, 14]. The establishment of a comparative karyotype between rat and mouse by cross-species chromosome painting allowed us to reconstruct more accurately the chromosome rearrangements sepa- rating both karyotypes and to define more precisely the homologous regions [15–17]. For this purpose, the painting probes were prepared directly from a low copy number of sorted chromosomes using PARM- PCR, afterwards, the mouse and rat specific painting probes were hybridized on rat and mouse metaphases, respectively. The availability of rodent species chromosome painting probes as well as the informations obtained by the comparative karyotype and comparative gene mapping data are of great interest to improve knowledge on species evolution. Moreover, these progresses could help to better understand carcino- genesis process by conducting cytogenetic analysis of tumors developed in either normal or transgenic animal models. Recently, multicolor painting, using combinatorial labeling of probes with five different fluorochromes, was applied on human and mouse metaphases [18–20]. Spectral karyotyping (SKY) and multi-fluor FISH (M-FISH) are based on this technique thus allowing to simultaneously identify each chromosome of a karyotype in distinct color. These technologies were applied to the cytogenetic characterization of numerous human lymphoid Methods in Cell Science 23: 163–170 (2001)  2001 Kluwer Academic Publishers. Printed in the Netherlands. Comparative karyotype using bidirectional chromosome painting: how and why? Marie-Noëlle Guilly, Laurent Dano, Patricia de Chamisso, Pierre Fouchet, Bernard Dutrillaux & Sylvie Chevillard CEA, DSV, DRR, Fontenay-aux-Roses, France Abstract. Rat is widely used in biomedical and pharmaceutical research but its genome has been significantly less studied than that of the mouse. This represents a major limitation for studying cytogenetic and molecular mechanisms in the rat model. As Muridae species underwent an intense chromosome evolution it is not possible to directly transpose knowledge of the mouse genome to that of the rat. For establishing a comparative karyotype between rat and mouse, painting probes of both species were prepared by PARM-PCR (Priming Authorizing Random Mismatches PCR) from a low copy number of sorted chromosomes, the mouse and rat specific painting probes being then hybridized on rat and mouse metaphases, respectively. The availability of rodent species chromosome painting probes as well Key words: Chromosome painting, Comparative cytogenetics, PARM-PCR amplification as the information obtained by the comparative karyotype and comparative gene mapping data are of great interest to improve knowledge on species evo- lution but also to better understand carcinogenesis process, as illustrated by our data concerning the cytogenetic characterization of radon-induced rat lung tumors. Detailed methods for obtaining painting probes by PARM-PCR from sorted mouse and rat chromosomes and for their hybridization in homol- ogous or heterologous conditions are described. Usefulness of chromosome painting is illustrated by the characterization of chromosomal abnormalities in a radon-induced rat lung tumor. Advantages and limitations of this technique as compared to classical cytogenetics, FISH and CGH are discussed.