The extent of purine nucleotide cycle (PNC) turnover [i.e., the cyclic deamination of adenosine 5'-monophosphate (AMP) and reamination of inosine 5'-monophosphate (IMP)] was estimated in fast-twitch muscle of pentobarbital sodium anesthetized rats during in situ stimulation, by the accumulation of excess IMP after blocking IMP reamination with the adenylosuccinate synthetase inhibitor, hadacidin. Sodium hadacidin (100 mg/kg, iv) did not alter IMP production and was effective in blocking IMP reamination by 80%. Hadacidin had no effect on IMP levels in muscle at rest or during mild stimulation (1 Hz for 30 min). During 30 min of more intense stimulation (5 Hz), hadacidin treatment resulted in a threefold increase in IMP in the fast-twitch white gastrocnemius section, but had no effect in the fast-twitch red section. The IMP in the fast-twitch white section did not accumulate over the 30-min period, but was produced initially (within 5 min) and kept for being reaminated by hadacidin. Thus, the IMP formation and IMP reamination evident in the saline-injected animals did not occur concurrently. This suggests that the IMP reamination arm of the PNC is not an important pathway in "working" muscle.