We have characterized the molecular forms of circulating insulins in patients with hyperinsulinemia of diverse etiology. We have also compared the efficacy of various chromatographic conditions using reversed-phase (RP) HPLC. Using 0.2% trifluoroacetic acid (TFA) and triethylamine (TEA) with acetonitrile as the organic modifier, at an elution rate of 0.17%/min, porcine, bovine, and human insulins could be easily separated as well as abnormal insulins in the plasma of a patient (J.R.) with hyperinsulinemia of unknown etiology. When the reversed-phase C18 column was changed and a gradient of 0.33%/min was used, the abnormal insulin in patient J.R. could not be separated. By changing the solvent system to acetonitrile and isopropanol (vol:vol, 3:1) containing 0.1% TFA, omitting the TEA, and using a gentle gradient of 0.1%/min, various semisynthetic analogues of human insulin could be easily separated and the abnormal insulin could be identified in the plasma of the patient J.R. Abnormal insulin was also found in a patient with MEN-I, but in contrast, the insulins in eight patients with benign sporadic insulinomas appeared to be normal. These results suggest that certain hyperinsulinemic states may be associated with an abnormal insulin and that RP-HPLC is useful for identification of insulin variants in the circulation. However, the conditions of RP-HPLC may be critical if the abnormalities of the insulin are subtle.