Alterations in the competency of the creatine kinase system elicit numerous structural and metabolic compensations, including changes in purine nucleotide metabolism. We evaluated molecular and kinetic changes in AMP deaminase from skeletal muscles of mice deficient in either cytosolic creatine kinase alone (M-CK-/-) or also deficient in mitochondrial creatine kinase (CK-/-) compared with wild type. We found that predominantly fast-twitch muscle, but not slow-twitch muscle, from both M-CK-/- and CK-/- mice had much lower AMP deaminase; the quantity of AMP deaminase detected by Western blot was correspondingly lower, whereas AMP deaminase-1 (AMPD1) gene expression was unchanged. Kinetic analysis of AMP deaminase from mixed muscle revealed negative cooperativity that was significantly greater in creatine kinase deficiencies. Treatment of AMP deaminase with acid phosphatase abolished negative cooperative behavior, indicating that a phosphorylation-dephosphorylation cycle may be important in the regulation of AMP deaminase.