We describe a direct, rapid, sensitive and highly specific radioimmunoassay for determining serum levels of 17-hydroxyprogesterone. It is based on the use of highly specific sheep antiserum, ¹²⁵I-labelled tracer, dextran-coated charcoal to separate the antibody bound and free fractions, and sodium salicylate to eliminate interference from endogenous binding proteins in serum. Intra- and interassay coefficients of variation are less than 8% and recovery is satisfactory. Sensitivity is 3.5 fmol per assay tube (0.14 nmol/l). Results correlate closely with those of an established technique using ³H-labelled steroid after initial solvent extraction and column chromatography of samples (y=1.02×−0.16; r=0.998). The values found for serum from normal adult subjects ranged from 1.0 to 12.1 nmol/l while those from treated and untreated patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency were 1.4–18.4 and 27.3–650 nmol/l, respectively.