Highly sensitive procedures for the characterization of molecular species of insulin in the circulation and for the isolation of the gene encoding human insulin have recently been developed. Nine subjects with abnormal forms of insulin or proinsulin have been reported. These include: [Leu B25], [Ser B24], [Leu A3] insulin as well as others that have not yet been identified. The clinical syndrome associated with the secretion of an abnormal insulin or proinsulin molecule presents with apparent endogenous insulin resistance with inappropriate high levels of insulin for the prevailing blood glucose concentration and a high insulin/C-peptide ratio due to the reduced catabolism of the abnormal insulin molecule. Diabetes occurs if there is concomitant insulin resistance or pancreatic beta cell failure. In addition, abnormal forms of insulin have been found in the insulin autoimmune syndrome presenting with recurrent, self-limiting hypoglycemia. Abnormal insulin in the autoimmune insulin syndrome suggests that abnormalities in endogenous antigens may be important in the formation of antibodies in other autoimmune states. However, although abnormalities in the insulin molecule may provoke the autoimmune response, it is also feasible that the presence of antibodies to normal insulin and proinsulin in some way alters their metabolism to yield abnormal products on high performance liquid chromatography (HPLC). Abnormal forms of insulin have also been found in subjects with reactive hypoglycemia and exogenous insulin-resistance and appear to be transmitted in an autosomal dominant manner. A protocol for the sequence of investigating subjects who potentially harbor a mutant or otherwise abnormal form of insulin using HPLC and recombinant DNA technology is presented.