The mechanism of dissociated secretion between adrenal androgens and cortisol observed in several clinical situations remains unclear. We investigated whether the electron transfer systems NADPH-cytochrome P450 reductase and cytochrome b5, both of which had been shown to increase 17,20-lyase activity in vitro, were involved in the reaction selectivity between 17 alpha-hydroxylase and 17,20-lyase in adrenocortical adenomas obtained from eight patients with Cushing's syndrome producing different concentrations of adrenal androgen. In vitro enzyme assay using microsomal fraction of adenoma indicated that all adenomas from seven patients showed almost the same degree of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activities. However, the 17,20-lyase activities of two adenomas producing high concentrations of adrenal androgens were 3-fold greater than those of other five adenomas producing low concentrations of adrenal androgens. The mRNA concentrations of cytochrome P45017 alpha and 3 beta HSD were approximately the same in all adenomas, whereas those of cytochrome b5 in two adenomas possessing high 17,20-lyase activities were greater than those in other adenomas. The increased levels of cytochrome b5 in the two adenomas were further confirmed at the protein level using Western blot analysis of the microsomal fraction. No significant expression of NADPH-cytochrome P450 reductase was detected in any of the adenomas by Northern blot analysis. These results suggest that the difference in the concentration of cytochrome b5 in adrenal adenomas from patients with Cushing's syndrome may partially account for the difference in the amount of adrenal androgens produced by the tumors.