In situ hybridization (ISH) of myosin heavy chain (MHC) mRNA, immunofluorescent detection of MHC protein, and oxidative enzyme histochemistry were performed on the same fibers in serially sectioned rabbit skeletal muscle. By combining these three techniques quantitatively, on a fiber-by-fiber basis, fibers that expressed mRNA complementary to a fast MHC cDNA pMHC24-79 of unknown subtype (Maeda et al., 1987) were classified into fiber types with respect to slow myosin expression and oxidative capacity. As expected, slow fibers had low hybridization to pMHC24-79. Fast fibers were divided into three subtypes. mRNA from the low oxidative fibers (fast-glycolytic, IIB) did not hybridize with pMHC24-79. Fast fibers whose mRNA hybridized best to pMHC24-79 were mainly in the intermediate range of oxidative capacity (probably IIX). The fast fibers with the highest oxidative capacity had low hybridization to this MHC mRNA (probably IIA). Thus, pMHC24-79 was identified as a clone of a fast isomyosin, tentatively designated as the fast IIX with intermediate oxidative capacity. The expression of more than a single species of fast and slow isomyosin mRNAs in classically defined fiber type was considered in interpreting these results.