Steroid Δ4-5α- and Δ4-5β-reductase activity was determined in 16 human mammary tumors and 8 DMBA-induced rat mammary tumors using a spectrophotometric assay. Steroid Δ4-5α-reductase was present in all tumors investigated while Δ4-5β-reductase was detected in only 6 estrogen receptor negative human breast tumors and absent in all estrogen receptor positive human breast tumors as well as in all rat mammary tumors. Further support for the presence of Δ4-5β-reductase was established by using a dual-labelling technique consisting of incubating tumor slices with [14C] testosterone and adding [3H] etiocholanolone, [3H] testosterone and [3H]-5α-dihydrotestosterone at the end of the reaction. Following extraction and chromic acid oxidation, 4-andro stenedione, 5β-androstanedione and 5α-androstanedione were isolated and purified, and the constancy of the 14C/3H ratio was used as proof of 5α-reductase and 5β-reductase. These results were shown to be consistent with the data obtained using the spectrophotometric assay.