Adenosine 5'-monophosphate (AMP) deamination, estimated from inosine 5'-monophosphate (IMP) accumulation, was studied in the different skeletal muscle fiber types of untrained rats anesthetized with ether immediately after 4 min of treadmill running at 45 or 60 m/min. The adenylosuccinate synthetase-inhibitor hadacidin was administered (200 mg/kg ip) before exercise to block IMP reamination and, therefore, to provide a better assessment of IMP formation. The increases in blood ammonia after exercise (2.5- and 5-fold, respectively) were highly correlated (r = 0.93) with the increases in blood lactate levels (6- and 11-fold). At both speeds, IMP increased in fast-twitch but not in slow-twitch (soleus) muscle. Of the fast muscles, the increase in IMP was greatest (up to 4 mumol/g wet wt) in the white vastus lateralis (fast twitch, glycolytic), intermediate in the plantaris (mixed fibers), and lowest in the red vastus lateralis (fast twitch, oxidative glycolytic). The increases in IMP were coincident with nearly equivalent decreases in ATP. Hadacidin treatment resulted in a greater IMP accumulation after exercise in both fast-twitch types but not in the soleus. The results indicate that fast-twitch muscle fibers, particularly the fast-twitch glycolytic fibers, are the source of the ammonia produced during strenuous exercise.