The contractile and associated metabolic characteristics of muscles are determined by their myosin heavy chain (MHC) isoform expression. In large mammals, the level of MHCIIB expression, which is associated with fast glycolytic-type muscle fibers, has not been fully characterized. In this study, quantitative reverse transcription-PCR and SDS-PAGE methodologies were developed for the analyses of adult ovine MHC isoform expression and used to characterize MHC expression in 3 skeletal muscles [LM, semitendinosus, and supraspinatus) from 66-d-old lambs. Three MHC isoforms (MHCI, MHCIIA, and MHCIIX) were detected at both the protein and messenger RNA levels in all 3 muscles, with greater proportions of type II than type I MHC. The expression of MHCIIB could not be detected at the protein level in any of the muscles and was detectable (in semitendinosus muscle) only at the messenger RNA level by using semiquantitative reverse transcription-PCR, indicating that MHCIIX is the predominant fast glycolytic fiber type in the sheep muscles studied. The methodologies developed are suitable for studying fiber type transformations at the molecular level, as well as allowing analyses of very small samples, including biopsies, when histochemical analysis may not be possible.