Abstract At radioimmunoassay (RIA) for 5α-androst-16-en-3α-ol in human and porcine plasma has been developed. Antibodies were produced in rabbits immunized against 5α-androst-16-en-3-(O-car☐ymethyl) oxime conjugated to BSA. The antiserum cross-reacted with other 16-androstenes as follows: 5α-androst-16-en-3α-ol, 42%; 4,16-androstadien-3-one, 23.8% 5α-androst-16-en-3β-ol, 16.1% and 5,16-androstadien-3β-ol, 1.19%. Although the method gave an acceptable standard curve for 5α-androst-16-en-3-one (10–1000 pg), it was not possible to separate by t.l.c. an unknown contaminant in plasma which interfered with the RIA of the steroid. However, by exploiting the high (42%) cross-reaction with 5α-androst-16-en-3α-ol, a RIA has been developed involving a thin layer chromatographic step. Regression analysis of the data in an accuracy study gave the equation y = 0.986 x + 151.5, with r = 0.999, while the coefficients of variation of replicate assays of plasma 5α-androst-16-en-3α-ol were in the range 9.5–15.6%. The mean values for plasma 5α-androst-16-en-3α-ol in 31 healthy men and 16 healthy women were 3.08 (range 0.3–14.9) and 0.66 (range 0–2.4) ng/ml, respectively. In boars, sows and castrated male pigs, the corresponding mean values were 30.7 (range 10.9–58.9), 0.24 (range 0.08–0.77) and 0.27 (range 0.06–0.51) ng/ml, respectively.