An antibody to androstanedione obtained in a rabbit by immunization with androstenedione-7 alpha-carboxymethyl-thioether conjugated to bovine serum albumin was found to cross-react 100% with 5 alpha-androstane-3,17-dione, a property that was used to develop a radioimmunoassay for this steroid. Plasma 5 alpha-androstane-3,17-dione concentrations were determined in young men, and in women throughout an ovulatory cycle. In the men (n = 6), plasma 5 alpha-androstane-3,17-dione concentrations were in the range of 84 to 273 pg/ml with a mean (+/- SD) value of 164 +/- 57 pg/ml. The plasma levels in the women (n = 5) were in the ranges of 35 +/- 14 to 145 +/- 75 pg/ml during the follicular phase, and 109 +/- 50 to 151 +/- 44 pg/ml during the luteal phase. The tissue sites of origin of 5 alpha-androstane-3,17-dione have not been defined, however, some extraglandular tissues are known to contain enzymes that convert C19-steroids to 5 alpha-androstane-3,17-dione. It is possible that 5 alpha-androstane-3,17-dione in circulation serves as a substrate for peripheral synthesis of 5 alpha-dihydrotestosterone.