We analyzed fiber types in rat skeletal muscles using a novel combination of in situ hybridization of myosin heavy chain (MyHC) mRNA, and enzyme histochemistry for succinate dehydrogenase (SD), which displayed metabolic properties. The fiber types were classified into the four major subtypes of I(beta/slow), IIA, IIX and IIB, and their intermediate types coexpressed two MyHC mRNAs: I and IIA, IIA and IIX, or IIX and IIB. The distribution of fiber types differed markedly in each skeletal muscle. The superficial region of limb muscles was composed mainly of fast-twitch fibers with oxidative-glycolytic and glycolytic activities, such as type IIX and type IIB. In contrast, the deep region was composed almost exclusively of type I and type IIA fibers both with oxidative activity. In this region, type IIA/IIX hybrid fibers were noted more frequently than type I/IIA and IIX/IIB hybrid fibers. In axial muscles, slow-twitch fibers and fast-twitch fibers composed predominantly of type IIB were distributed dispersively. The diaphragm and masseter showed a high proportion of type IIX and type IIB, respectively, to adapt to tissue-specific functional requirements and more frequently contained type IIX/IIB hybrid fibers than other observed muscles.