A radioimmunoassay for plasma 3 beta, 7 alpha-dihydroxy-5-androsten-17-one (7 alpha-hydroxy DHA) has been developed using anti-sera raised against 3 beta, 7 alpha-dihydroxy-5-androstene-17 beta-carboxyl-bovine serum albumin conjugate and [1, 2 (n) - 3H] 7 alpha-hydroxy DHA as the radioligand. Significant cross reactivity was found with 3 beta, 7 alpha-dihydroxy-5-pregnen-20-one (44%), 3 beta, 7 beta-dihydroxy-5-androsten-17-one (6%), 3 beta, 6 beta-dihydroxy-4-androsten-17-one (2.5%), 3 beta-hydroxy-5-androsten-17-one (DHA, 2%), 3 beta, 7 beta-dihydroxy-5-pregnen-20-one (2%) and 7 alpha-hydroxy-4-androstene-3, 20-dione (1%). 7 alpha-Hydroxy DHA was extracted from plasma and separated from cross-reacting factors using alumina micro-columns. The separation of bound and free steroid was achieved using dextran-coated charcoal. The concentration of 7 alpha-hydroxy DHA in the plasma of breast cancer patients was significantly lower than the concentrations in the plasma of normal women, hospitalized women suffering from non-endocrine diseases and patients with benign breast disease. The decrease in the concentration of 7 alpha-hydroxy DHA in the plasma of pregnant women was not significant.