We have developed a reliable radioimmunoassay for testosterone in plasma, polyethylene glycol ("Carbowax 6000") being used to separate antibody-bound and free hormone. Testosterone is separated from interfering steroids, notably dihydrotestosterone, by liquid—liquid partition chromatography on infusorial earth (Celite). The assay is sensitive (9 pg for standards), precise, and accurate. The lowest measurable concentration of testosterone is 350 ng/liter for plasma from men and 70 ng/liter for plasma from women. Intraand inter-assay coefficients of variation were 6.9% and 9.7%, respectively, for plasma from men, and 9.6% and 11.8%, respectively, for plasma from women. Our method for separating antibody-bound and free hormone is practical and convenient and may be generally applicable to all radioimmunoassays of steroid hormones in plasma.