We investigated the expression of neuronal nitric oxide synthase (nNOS) and dystrophin in the regenerating skeletal muscles of rats after cardiotoxin-induced myonecrosis by immunohistochemical studies and western blot analysis. In normal muscles, nNOS was moderately immunostained on type 2B fibers, but was faintly immunostained on type 2A or type 1 fibers. In immunohistochemical studies of regenerating muscles, nNOS was first observed at the sarcolemma of type 2B fibers on day 10, when the type discrimination between types 2A and 2B was first detected by ATP reactions. Subsequently, the immunostaining of nNOS grew progressively stronger in type 2B fibers, with faint staining in type 2A and type I fibers until day 28. Meanwhile, the immunostaining of dystrophin grew stronger equally in all three fibers until day 21. In western blot analysis of regenerating muscles, nNOS regenerated more slowly than dystrophin. The present data suggest that the expression of nNOS is related to the muscle fiber type differentiation, and that the role of nNOS is related to the function of the type 2B fibers of the muscle.