Human serum contains a very potent inhibitor of creatine kinase in addition to urate which was characterized as an inhibitor of this enzyme by Warren (Warren, W.A. (1975) Clin. Biochem. 8, 247). This new inhibitor was isolated from human serum by dialysis, negative DEAE-cellulose adsorption and a series of gel filtration chromatographies. The inhibitor so isolated was an organic compound. Kinetic investigation showed noncompetitive inhibition versus creatine, as well as MgATP. Infrared spectroscopy revealed amino and carboxyl groups. The isolated material was ninhydrin-positive. Thin-layer chromatography of the dansyl derivative showed several fluorescent spots. Quantitative amino acid analysis revealed the presence of several amino acids. Of these only cystine and cysteine were inhibitory in the standard inhibitor assay, using rabbit MM creatine kinase. Testing with human MM creatine kinase only cystine was found inhibitory. Cystine has a specific inhibitor activity of 600 anti-Units per mg, using rabbit or human MM creatine kinase, respectively. Urate by comparison has a specific inhibitor activity of 45 anti-Units per mg using human MM creatine kinase.