This rapid, inexpensive, and sensitive radioimmunoassay (RIA) for plasma 17 alpha-hydroxyprogesterone involves radioiodination. A single extraction with toluene/hexane removes an average 93% of the hormone from 0.1 mL of plasma. The extract is evaporated and the hormone is estimated by a simple, precise, and accurate 125I RIA involving a specific rabbit antiserum. A suspension of dextran-coated charcoal is used to separate free and bound steroid. Inter- and intra-assay CVs were less than 15 and less than 10%, respectively, and the sensitivity was 3 pg per assay tube. The regression equation for data on 17 alpha-hydroxyprogesterone added to steroid-free plasmas was y = 0.94x + 2.2 (r = 0.99). However, the turnaround time is only one-half to one-tenth that for most 3H RIA (3 h vs 6 to 30 h). The ranges of values found for plasma from normal subjects, treated and untreated patients with congenital adrenal hyperplasia, and infants with newly detected congenital adrenal hyperplasia were, respectively, 1 to 11, 0 to 20, 30 to 620, and 270 to 4900 nmol/L.