A double isotope derivative technique using 14C indicator and 3H-acetic anhydride to estimate testosterone, androstenedione, dehydroepiandrosterone, androsterone, etiocholanolone and 11β3-OH-Δ4-androstenedione in peripheral, adrenal and gonadal venous plasma is described. With this method, plasma from ovariectomized-adrenalectomized or hypophysectomized-adrenalectomized-gonadectomized subjects gives values of 0.0043±0.0019 (sd) for T, 0.0024±0.0027 (sd) for D, 0.0031±0.0021 (sd) for Δ, 0.0036±0.0026 (sd) for E, and 0.0047±0.0013 (sd) for A in μg/100 ml. Water blanks give values of 0.0017±0.0009 (sd) for T, 0.0021±0.0011 (sd) for Δ, 0.0014±0.0007 (sd) for D, 0.0026±0.0018 (sd) for E, 0.0020±0.0012 (sd) and 0.0020±0.001 (sd) for 11βOH-Δ μg/100 ml. Mean peripheral plasma values for these steroids in normal men are: 0.67±0.23 (sd)±0.29 (SE) for T, 0.51±0.29 (sd)±0.039 (SE) for D, 0.13±0.04 (sd)±0.006 (SE) for Δ, 0.07±0.06 (sd)±0.014 (SE) for E, 0.16±0.08 (sd)±0.019 (SE) for A and 0.20±0.08 (sd)±0.021 (SE) for 11β-OH-Δ μg/100 ml. Peripheral plasma values are also cited for old men with and without gonads; normal, oophorectomized and postmenopausal women; women with idiopathic hirsutism; normal children and patients with adrenal disorders and those with Turner's syndrome. Concentrations of these steroids in gonadal and adrenal venous effluent are also reported.