Metabolic control within skeletal muscle is designed to limit ADP accumulation even during conditions where ATP demand is out of balance with ATP synthesis. This is accomplished by the reactions of adenylate kinase (AK; ADP+ADP <--> AMP+ATP) and AMP deaminase (AMP+H(2)O --> NH(3)+IMP), which limit ADP accumulation under these conditions. The purpose of this study was to determine whether AK deficiency (AK(-/-)) would result in sufficient ADP accumulation to be visible using (31)P-NMRS during the high energy demands of frequent in situ tetanic contractions. To do this we examined the high-energy phosphates of the gastrocnemius muscle in the knockout mouse with AK1(-/-) and wild-type (WT) control muscle over the course of 64 rapid (2/s) isometric tetanic contractions. Near-complete depletion of phosphocreatine was apparent after 16 contractions in both groups. By approximately 40 contractions, ADP was clearly visible in AK1(-/-) muscle. This transient concentration of the NMR visible free ADP was estimated to be approximately 1.7 mM, and represents the first time free ADP has been directly measured in contracting skeletal muscle. Such an increase in free ADP is severalfold greater than previously thought to occur. This large accumulation of free ADP also represents a significant reduction in energy available from ATP, and has implications on cellular processes that depend on a high yield of energy from ATP such as calcium sequestration. Remarkably, the AK1(-/-) and WT muscles exhibited similar fatigue profiles. Our findings suggest that skeletal muscle is surprisingly tolerant to a large increase in ADP and by extension, a decline in energy from ATP.