We investigated the onset and duration of DNA synthesis and differentiation of muscle precursor cells in the early stage in muscle regeneration after bupivacaine induced muscle necrosis using monoclonal anti-PCA and anti-desmin antibodies. The first positive PCNA reaction indicating the commencement of proliferating cycle of muscle precursor cells appeared at 24 hours after injury, and were most prominent at 60-72 hours after injury. Thereafter the reaction for PCNA disappeared as the maturation of regenerated myofibers progressed. Some nuclei of regenerated myotubes in the early stage were positive for PCNA contrary to the generally accepted concept that myonuclei after fusion were out of cell cycle. The first desmin expression was observed in mononuclear presumptive myoblasts at 48 hours after injury, some of which were also positive for PCNA in the double immunostained sections for desmin and PCNA. This result suggested that differentiation of presumptive myoblasts could already initiate during their proliferative phase.