A specific and simple radioimmunoassay was developed for human proinsulin. Antiserum against human proinsulin was produced in guinea pigs immunized with an emulsion of antigen and Freund's adjuvant. Only one out of six guinea pigs produced proinsulin-specific antiserum that did not crossreact with human insulin or C-peptide. A double antibody method, using anti-guinea pig γ-globulin rabbit serum as a second antibody, was used to separate bound and free ¹²⁵I-proinsulin in the reaction mixture. With this radioimmunoassay, it was possible to quantify directly without extracion 0.003 pmol/ml of circulating human proinsulinin serum. The mid-range (IC 50) of the assay was 0.075 pmol/ml. The mean intra-and interassay coefficients of variation were 5.5% and 9.8%. The recoveries of proinsulin added to serum were in the range 92-119%. The normal fasting proinsulin level in serum was 0.008±0.003pmol/ml, increasing gradually after a 100-g oral glucose load, and reached a peak level of 0.021±0.006 pmol/ml after 120 min.