This report describes the simultaneous radioimmunoassay of plasma testosterone (T) and 5a-di hydrotestosterone (DHT), using as binding reagent a highly specific antiserum which reacted significantly with T and DHT. Utilizing a one step celite microcolumn to separate T from DHT, we have measured these two androgens accurately in small aliquots of plasma from both male and female subjects. Plasma concentrations of T and DHT expressed as mean ± S.D. in ng/ml were respectively: 1) in male subjects: a. normal adult: 4.9 ± 1.6 (T) and 1.310.8 (DHT); b. hypogonadal: 0.24 ± 0.08 (T) and 0.20 ± 0.10 (DHT); c. prepubertal: 0.14 ± 0.02 (T) and 0.05 ± 0.02 (DHT). 2) in female subjects: a. normally menstruating, irrespective of time of cycle: 0.22 + 0.07 (T) and 0.34 ± 0.17 (DHT); b. postmenopausal: 0.29 ± 0.08 (T) and 0.15 ± 0.07 (DHT); c. ovariectomized: 0.24 ± 0.06 (T) and 0.09 ± 0.01 (DHT). When plasma levels of T and DHT were measured during a complete menstrual cycle, the levels of these steroids were found to be higher during the luteal than during the follicular phase. The ratio of plasma TsDHT concentrations was five times higher in normal adult male (3.1) than in menstruating female subjects (0.6). © 1972, Taylor & Francis Group, LLC. All rights reserved.