We investigated the effects of sulfhydryl compounds on the stability of creatine kinase (CK) in unfrozen human serum and found that both β-mercaptoethanol and N-acetylcysteine led to accelerated loss of the endogenous serum enzyme activity. This is in contrast to the results of others who have either studied the stability of exogenous enzyme added to human serum or studied endogenous enzyme in frozen serum. The addition of cation chelators to serum markedly improved the stability of the endogenous CK activity. The enhanced stability was independent of chelation of calcium, iron, manganese, copper, or zinc. In addition, cation chelators caused a 16% increase in the CK activity of fresh samples. This latter effect was independent of the activation of CK by BME and could be accounted for by chelation of calcium ions during the assay. The data suggest that addition of cation chelators prior to storage may be useful in enhancing the stability of CK in serum whereas sulfhydryl compounds should be added prior to assay rather than prior to storage.