The details of a radioimmunoassay capable of measuring as 5 pg of prostaglandin A, E, and F (PGA, PGE, and PGF) in human and rat plasma are described. Plasma samples are extracted (with 4000 cpm [(3)H] PGE(1) added for calculation of recovery) with an organic solvent system at an apparent pH of 5.8 and then chromatographed on silicic acid columns with increasing concentrations of methanol to separate PGA, PGE, and PGF. Each chromatographed sample is measured by radioimmunoassay, using the homologous antibody and tritiated marker. 40 normal individuals had mean plasma concentrations of PGA, PGE, and PGF of 1062+/-107 pg/ml, 385+/-30 pg/ml, and 141+/-15 pg/ml, respectively. Elevated PGE levels were measured in the plasma of patients with medullary carcinoma of the thyroid, carcinoid, and neuroblastoma. Treatment of rats with indomethacin decreased serum PGE levels by 67%. The radioimmunoassay appears to be of considerable experimental as well as clinical interest.