Seven unconjugated neutral steroids, including testosterone and some of its precursors and metabolites, were measured in the peripheral and spermatic venous blood males, employing specific radioimmunoassays after the fractionation of steroids on Lipidex-5000 (hydroxyalkoxypropyl Sephadex) microcolumns. Respective mean concentrations (ng/ml) and ranges of steroids estimated in peripheral and spermatic venous blood in all groups of patients were as follows: pregnenolone, 0.71 (0.29-2.39) and 10.97 (0.83-30.1); progesterone, 0.31 (0.02-0.57) and 10.17 (1.51-33.24); 17 alpha-hydroxyprogesterone, 1.04 (0.48-2.20) and 37.33 (1.68-141.00); androstenedione, 1.01 (0.26-2.65) and 11.87 (0.97-30.18); testosterone, 3.84 (0.63-10.64) and 255.1 (2.85-619.1); 5alpha-dihydrotestosterone, 0.19 (0.07-0.28) and 3.74 (0.04-9.71); androsterone, 0.27 (0.12-0.47) and 0.97 (0.20-2.15). Concentrations are similar to those estimated by mass spectrometry and protein binding assays, except for androsterone which has not previously been measured in this context. The low, but significant testicular secretion of both 5alpha-hydrotestosterone and androsterone suggests that these two steroids are testicular androgen metabolites, and that androgen metabolism in this tissue may be monitored by way of their measurement in spermatic vein blood.