A simplified colorimetric assay for serum ATP: creatine phosphotransferase activity is described and factors affecting enzymatic and colour reactions outlined. The method incorporates a sulphydryl reagent and eliminates the need for protein precipitation. Estimations in 519 normal subjects demonstrated higher values in males than in females and showed a significant correlation with age and with body weight. Normal values were found in 5% of established cases of myocardial infarction and raised values in 10% of cases in whom this diagnosis was initially entertained but ultimately excluded; diagnostic accuracy compared favourably with that of spectrophotometric aminotransferase determinations in the same patients. Results by the present method agreed well with those obtained by an established technique in cases of muscle disease and relatives of affected subjects. Activity in the cerebrospinal fluid of patients with a variety of neurological diseases was barely detectable and of no diagnostic value. Study of serum dilution, and of the interaction of native, heated, and dialysed sera, with rabbit muscle enzyme, suggested the presence of two inhibitors of creatine phosphotransferase activity in human serum.