Homocysteine thiolactone was isolated from normal human plasma, sera of patients with acute myocardial infarction, and human neoplastic tissue. The compound was identified by ninhydrin reaction and by nitroprusside reaction following alkaline hydrolysis. Thin layer and ion exchange chromatography demonstrated a single uniform peak which co-chromatographed with authentic homocysteine thiolactone. The substance was isolated by extraction of plasma, serum or tissue with chloroform:methanol, re-extraction of the chloroform layer with dilue HCl, and evaporation of the resulting aqueous acid layer. The amount of homocysteine thiolactone was found to be 1,670 mumol/L in normal human serum and 12,500 mumol/L in sera of coronary heart disease patients. Although there is variability and overlap of values between groups, the difference between the two groups is significant (P = 0.025). The amount of homocysteine thiolactone in three human neoplasms was 17-123 mumol/kg. Only trace amounts were found in normal tissues, and none was detected in two other human neoplasms. The findings support the previous demonstration of abnormal homocysteine thiolactone metabolism in arteriosclerosis and cancer.