&p.1:Abstract Combined methodologies of histochemistry, immunohistochemistry, sodium dodecyl sulphate poly- acrylamide gel electrophoresis (SDS-PAGE), reverse transcriptase polymerase chain reaction (RT-PCR) and a histochemical method specific for myofibrillar ATPase (mATPase) of the type IIX myosin heavy chain (MyHC) isoform were used to study human and rat sin- gle fibres to examine the homology between type II MyHC isoform-based fibres of both species. We dem- onstrate that human type II fibres exhibit antigenic mATPase and 3’-untranslated region (3’-UTR) se- quence determinants homologous to the IIA and IIX but not the IIB MyHC isoforms of the rat. Both immunola- belling with anti-MyHC monoclonal antibodies and the mATPase method used with frozen sections confirmed that all human type II fibres express type IIA and/or type IIX MyHC. Quantitative immunohistochemistry failed to recognize human fibres with antigenic charac- teristics corresponding to hybrid IIXB MyHC-based fi- bres. Ca 2+ -stimulated maximum myosin ATPase activi- ty, determined by quantitative histochemistry, revealed that human IIX fibres (with an optical density or OD = 0.707) display enzyme activity which is compa- rable to that of the rat type IIX (OD = 0.687) but lower than that of the rat type IIB fibres (OD = 0.836). The results do not support the notion that MyHC IIB is ex- pressed in human limb muscles, even in hybrid fibres. We conclude that human type II fibres have been mis- classified in numerous previous publications and that this has important implications in attempts to compare the physiological characteristics of fibre types, particu- larly when animal models are used. &kwd:Key words Myosin heavy chain isoforms · Histochemical analysis · Immunohistochemical analysis · Fibre type classification&bdy: Introduction Our understanding of sarcomeric myosin polymorphism in mammalian skeletal muscle has been extended by the recognition that more types of myosin exist than the types I, IIA and IIB indicated by ATPase histochemical meth- ods based on acid and alkaline denaturation [23]. A third fast skeletal muscle myosin heavy chain (MyHC) isoform was initially designated as either IIX [22] or IID [2]. Al- though it is well documented that this isoform is widely expressed in muscles of smaller mammals [9] its expres- sion in human muscle has only recently been reported [11, 25]. The combination of molecular and immuno- chemical characterization of single fibres [11] and whole- muscle samples [25] suggests that human skeletal mus- cles may not express a MyHC isoform homologous to type IIB which is expressed in the rat and other small mammals. Although these observations appear to be con- sistent with the fact that many laboratories have failed to recognize expression of a type IIB or a IIB-like MyHC gene in human skeletal muscle, they disagree with earlier immunoblotting studies reporting the presence of a hu- man MyHC isoform equivalent to the rat type IIB [16]. Because molecular genetic analysis suggests that the cat- alogue of human MyHC isoforms is still incomplete [33], J.A.A. Sant’Ana Pereira 1 · S. Ennion · G. Goldspink ( ✉ ) Department of Anatomy and Developmental Biology, The Royal Free Hospital School of Medicine, Division of Biomedical Sciences, Rowland Hill Street, Hampstead, NW3 2PF, London, UK J.A.A. Sant’Ana Pereira · A.F.M. Moorman Department of Anatomy and Embryology, Academic Medical Centre, University of Amsterdam, The Netherlands J.A.A. Sant’Ana Pereira · A.J. Sargeant Neuromuscular Biology Unit, Manchester Metropolitan University, Manchester, UK S. Ennion Department of Geriatric Medicine, The Royal Free Hospital School of Medicine, University of London, UK 1 Present address: University of Wisconsin Medical School, Department of Physiology, 122 Service Memorial Institute, 1300 University Avenue, Madison, WI 53706, USA&/fn-block: Pflügers Arch – Eur J Physiol (1997) 435:151–163 © Springer-Verlag 1997 ORIGINAL ARTICLE &roles:José A.A. Sant’Ana Pereira · Steven Ennion Anthony J. Sargeant · Antoon F.M. Moorman Geoffrey Goldspink Comparison of the molecular, antigenic and ATPase determinants of fast myosin heavy chains in rat and human: a single-fibre study &misc:Received: 15 April 1997 / Received after revision: 9 July 1997 / Accepted: 21 July 1997