A method is described for the determination of 17 alpha-hydroxyprogesterone (17-OHP) in plasma. Antisera were raised in rabbits against 11-deoxycortisol-21-hemisuccinate-bovine serum albumin (11-DOC-21-HS-BSA) and 17 alpha-hydroxyprogesterone-3-carboxymethyloxime-bovine serum albumin (17-OHP-3-CMO-BSA). An antiserum to 11-DOC-21-HS-BSA exhibited cross-reaction with progesterone (29%), 11-deoxycortisol (100%), cortisol (17%) and testosterone (10%) and was therefore not appropriate for quantitation of 17-OHP in plasma. An antiserum to 17-OHP-3-CMO-BSA cross-reacted with progesterone (9,7%), 11-deoxycortisol (50%) and less than 1% with all other major naturally occurring steroid hormones. A radio-immunoassay (RIA) was developed using the antiserum to 17-OHP-4-CMO-BSA. The intra-assay and interassay coefficients of variation were 7,2% and 10,5% respectively. The normal ranges (nmol/l plasma) of samples extracted with hexane:benzene (1:1) and purified by Sephadex LH-20 chromatography were 0,28-4,7 for men, 0,84-3,0 for women (follicular phase), 3,0-11,0 for women (luteal phase), 4,6-22,1 for pregnant women, 18,5-123,9 for cord blood, 0,12-5,0 for children and 56,3-1 032 for persons with congenital adrenal hyperplasia (CAH) due to a 21-hydroxylation enzyme defect. Sephadex LH-20 purification of plasma extracts could be omitted when using the RIA as a screening procedure for CAH due to a 21-hydroxylation enzyme defect.