The study of cellular energetics is critically dependent on accurate measurement of high-energy phosphates. Muscle values of phosphocreatine (PCr) vary greatly between in vivo measurements (i.e., by nuclear magnetic resonance) and chemical measurements determined from muscles isolated and quick-frozen. The source of this difference has not been experimentally identified. A likely cause is activation of ATPases and phosphotransfer from PCr to ADP. Therefore, rat hindlimb skeletal muscle was perfused either with or without 2 mM iodoacetamide, a creatine kinase inhibitor, and muscle was freeze-clamped either at rest or after contraction. Creatine kinase inhibition resulted in approximately 6 micromol/g higher PCr and lower creatine in the freeze-clamped soleus, red gastrocnemius, and white gastrocnemius. This PCr content difference was reduced when the initial PCr content was decreased with prior contractions. Therefore, the amount of PCr artifact appears to scale with initial PCr content within a fiber-type section. This artifact directly affects the measurement and, thus, the calculations of muscle energetic parameters from studies using isolated and frozen muscle.