The differentiation of fiber types in normal and neonatally denervated gastrocnemius muscles of the rat was compared by myosin ATPase histochemistry and immunocytochemistry using a monoclonal antibody, HM-1.2. The specificity of HM-1.2 for the fast myosin heavy chain was determined by radioimmunoassay, immunoautoradiography, and indirect immunofluorescence techniques. In normal 1-month-old and adult rats, the type IIB (fast glycolytic) fibers of the gastrocnemius could be clearly divided into three subtypes by their graded immunofluorescence staining with the myosin heavy chain-specific monoclonal antibody. In the gastrocnemius muscle of the newborn rat, all fibers were negative with the monoclonal antibody. The transition from negative to three grades of immunoreactivity occurred 1 to 2 weeks postnatally. After neonatal denervation of the gastrocnemius muscle, however, uniformly positive monoclonal antibody immunofluorescence staining for the myosin heavy chain was observed without subtype differentiation. This study, thus, gave clear immunocytochemical evidence that the type IIB muscle fibers are heterogeneous with respect to their myosin isoform and that the expression of this heterogeneity is dependent on the normal developmental influence of motor innervation on the muscle fibers.