Freely grafted rat extensor digitorum longus (EDL) muscles were subjected to low-frequency stimulation in an anaerobic environment to determine whether regenerating fast-twitch muscles regain normal glycolytic metabolic capacity. Regenerating muscles were tested at 28, 42, and 76 days after the graft procedure. Stabilized grafts (76 days) produced approximately 60% of the lactate generated by intact, control EDL subjected to the same stimulus paradigm and developed half the estimated increase in H+. The grafts exhibited the same relative decline in force after 5 min of anaerobic stimulation as control EDL but maintained relatively constant levels of ATP while consuming phosphocreatine. This study indicates that regenerating fast-twitch skeletal muscle has a reduced ability to initiate glycolytic activity during exercise. The data also indicate that a small population of regenerating fast-twitch fibers express the slow isoform of myosin heavy chain (beta-MHC) with maximum expression occurring at 56 days postsurgery.