Fifty-nine samples taken from lean and obese healthy subjects and from patients with diabetes mellitus, myotonic dystrophy, and islet cell adenoma after the administration of oral or intravenous glucose or other insulin secretogogues were fractionated on Sephadex G-50 columns. Eluates corresponding to zones of elutions of porcine pro insulin (“big insulin”) and Regular insulin were assayed for insulin by radioimmunoassay using crystalline human insulin as standard and porcine insulin-I-125 as tracer. In four samples from one patient with islet cell adenoma the big insulin fraction composed 24 to 55 per cent of the apparent insulinimmunoreactivity but in no other case did big insulin exceed 20 per cent of the total and in fifty-one of the fifty-five samples other than from the patient with insulinoma, big insulin contributed less than 15 per cent to the total apparent immunoreactive plasma insulin.