Since the advent of radioimmunoassay, I the concentration of insulin in plasma of normal and diabetic subjects in response to many stimuli has been studied extensively.2 However, the chemical nature of the circulating hormone is not entirely clear. We find that endogenous plasma insulin has two components.One is very similar to pancreatic insulin. The other component is larger in size and immunologically less reactive than pancreatic insulin. With our antiserum, it accounts for up to 50 per cent of plasma insulin measured by immunoassay.Materials and Methods.-Purified crystalline porcine and bovine insulin were gifts of Eli Lilly. Highly purified A-chain and B-chain sulfonates of bovine insulin were gifts of Dr. Arnold Marglin.' Cytochrome c, pancreatic ribonuclease, and porcine adrenocorticotropin (ACTH) were purchased from Mann, crystalline human serum albumin from Armour, rabbit fraction II and normal guinea pig serum from Pentex, guinea pig fraction II from Hyland, and NaI'25 and NaI"11 from Union Carbide. Guinea pig antiporcine insulin serum was a gift of Drs. A. Kagan and S. M. Glick. Antibody to guinea pig gamma globulin was generated in sheep by repeated injection of guinea pig fraction II in complete Freund's adjuvant (Difco). Albumin and ACTH were labeled with 112 and insulin with I'll by the chloramine T method at specific activities of 5, 5, and 450 uc/igg, respectively.4 The albumin-I125 was freed of radioactive contaminants by batch adsorption with Dowex 1X10. The ACTH-I'25 was purified by adsorption to silica and elution with acetic acid and acetone.' The insulin-I131, prepared fresh for each assay, was purified on a cellulose column.1 The normal subjects for the experiments were healthy male volunteers in their early twenties without a family history of diabetes. For the standard oral glucose tolerance tests 100 gm of glucose was administered in the morning after an overnight fast; for 3 days prior to the studies the subjects received a normal diet that included 300 gm of carbohydrate daily. For the starvation tests, food was withheld until the urine reacted strongly positive for acetone; the fast, about 36 hr in duration, was terminated by the administration of glucose, 100 gm orally. Patient MIT is an obese adult female with long-standing glucose intolerance, delayed hyperinsulinism following oral glucose, and retinal microaneurysms typical of diabetes. Plasma that was essentially free of insulin was obtained after a 72-hr fast from a patient who had had an islet-cell adenoma removed several years earlier. None of the subjects had ever been treated with insulin.Blood was drawn into heparin, refrigerated, and centrifuged, and the plasma stored at -20°. Plasma, 1-3 ml, was enriched with albumin-I125 and iodidel25 and applied to a 1 X 50-cm column of Sephadex G-50 fine (Pharmacia). Shorter columns or coarser grades of gel were unsatisfactory. Since the results of radioimmunoassay can be affected by variations in the salt content of the samples,' the column was equilibrated and developed in the same...