This study was conducted to evaluate further the reaction catalyzed by the saturated steroid 6alpha-hydroxylase of extrahepatic human tissues. Progesterone and 5alpha-dihydroprogesterone (5alpha-DHP) are plasma-borne precursors of 5alpha-pregnan-3alpha-ol-20-one, an anxiolytic/anesthetic steroid, and 5alpha-pregnan-3beta-ol-20-one in extrahepatic human tissues. These two steroids are metabolized further by a saturated steroid 6alpha-hydroxylase enzyme(s) that is distinct from the cytochrome P450 6alpha-hydroxylase that catalyzes the 6alpha-hydroxylation of delta4-3-ketosteroids such as progesterone, cortisol, and testosterone. Products of this saturated steroid 6alpha-hydroxylase, viz. 3beta/alpha,6alpha-dihydroxy-5alpha-pregnan-20-ones, are major radiolabeled urinary metabolites (excreted as glucuronosides) of i.v. administered tritium-labeled 5alpha-DHP in women and men. T47-D human breast cancer cells, which are rich in saturated steroid 6alpha-hydroxylase activity, were used as the enzyme source in this study. The greatest total and the highest specific activity of saturated steroid 6alpha-hydroxylase were localized in microsome-enriched preparations; enzyme activity was linear with incubation time up to 30 min and with microsome-enriched tissue protein concentrations between 0.05-0.5 mg/mL incubation mixture. The velocity of the reaction was similar in incubations in which the pH was varied from 6.0-8.0, and NADH and NADPH were equally effective in supporting the 6alpha-hydroxylation of 5alpha-pregnan-3beta-ol-20-one and 5alpha-pregnan-3alpha-ol-20-one. The more efficient substrates for this enzyme were 5alpha-pregnan-3beta-ol-20-one and 5alpha-pregnan-3alpha-ol-20-one, and the apparent Km (approximately 3.5 micromol/L) and maximum velocity (approximately 150 pmol/min x mg microsome-enriched protein) for these two substrates were indistinguishable. 5alpha-Androstane-3beta,17beta-diol was less efficiently 6alpha-hydroxylated, and 5alpha-androstane-3alpha,17beta-diol was an inefficient substrate. The addition of a variety of inhibitors of cytochrome P450 monooxygenases to the incubation mixtures did not diminish significantly the 6alpha-hydroxylation of 5alpha-pregnan-3beta-ol-20-one, findings consistent with those of other investigators who suggested that human saturated steroid 6alpha-hydroxylase (of human prostate) is not a cytochrome P450.