The rate of AMP deamination to IMP and NH4, by the action of AMP deaminase, is increased in vitro by acidosis and elevations in [AMP] and [ADP]. We evaluated the influence of acidosis on the activity of AMP deaminase in contracting muscle (5 Hz) by relating the time course of IMP and NH4 production to lactate-induced acidosis in low-oxidative, fast-twitch white (FTW) and high-oxidative, fast-twitch red (FTR) muscle of the rat. Cellular acidosis was modified by controlling lactic acid accumulation by regulating muscle blood flow and using trained animals. A significant activation of AMP deaminase occurred in both muscle types, but only at times when the estimated pH was 6.6 and below (lactate content 20 mu mol/g and above). Cellular acidosis, however, is not absolutely essential, since iodoacetic acid-blocked muscle lost 85-90% of its ATP to IMP during contractions. Thus cellular acidosis seems to be an important, but not the sole, factor activating AMP deaminase during contractions. Further, the influence of acidosis is probably different between fiber types, since the estimated free AMP and ADP contents, calculated from the creatine kinase and myokinase reactions, were different in the two fiber types. Most of the activation of AMP deaminase in FTR muscle could be attributed to a substrate effect of the increased free AMP content. In contrast, most of the activation of AMP deaminase in the FTW muscle was due to factors other than a substrate effect. These results suggest that cellular acidosis during intense contraction conditions is a major factor activating AMP deaminase, especially in the low-oxidative FTW muscle fiber type.