Several members of the sarcomeric myosin heavy chain (MHC) gene family have been mapped in the human genome but many of them have not yet been identified. In this study we report the identification of two human skeletal MHC genes as fast IIa and IIx MHC based on pattern of expression and sequence homology with the corresponding rat genes in the 3'-translated and untranslated regions. The distribution of these two gene products as well as that of the beta/slow MHC gene was analyzed in human skeletal muscles by in situ hybridization. The distribution of beta/slow, IIa, and IIx MHC transcripts defines three major muscle fiber types expressing a single MHC mRNA, i.e., either beta/slow, IIa, or IIx MHC mRNA, and two populations of hybrid fibers coexpressing beta/slow with IIa or IIa with IIx MHC mRNA. Fiber typing by ATPase histochemistry shows that IIa MHC transcripts are more abundant in histochemical type IIa fibers, whereas IIx MHC transcripts are more abundant in histochemical type IIb fibers.