A specific radioimmunoassay has been developed for diethylstilbestrol (DES), using an antiserum raised against DES monocarboxymethyl ether and a tritium-labeled radioligand. Prior to radioimmunoassay, a fraction enriched in DES is ob tained from a dichloroethane extract of plasma using Sephadex LH-20. The specificity of the assay is good, and the sensitivity (130 pg/ml) is adequate for accurate determination of DES in plasma from prostatic cancer patients treated with the drug. The precision is satisfactory, with an interassay coefficient of variation of approximately 10% at concentrations of approxi mately 1 ng/ml, and the blank values are negligible. Excellent agreement (r = 0.96) is observed between data obtained by radioimmunoassay and those obtained by a procedure using gas chromatography-high-resolution mass spectrometry. DES concentrations in the plasma of six treated (1 mg DES three times daily) patients were in the range 0.15 to 6.0 ng/ml. Increases in plasma concentration were observed within 2 hr of administration, with secondary rises occurring 5 to 6 hr later. Plasma testosterone concentrations were low in four of the patients; in a single subject, relatively high levels of testoster one were further elevated following administration of luteinizing hormone-releasing hormone.