Normal adrenal and adrenal tumor cells from a female infant with a virilizing adrenal tumor were grown in tissue culture as monolayers for a period of 7 weeks. Half of the cultures were exposed to ACTH (0.1 U/ml). The cells grew well and continued to produce steroid hormones over the entire period in culture. Production of steroid hormones was measured by RIA of individual steroids in the culture medium. Unstimulated normal and tumor cells produced equivalent amounts of cortisol, 11 beta, 18, 21-trihydroxy-4-pregnene-3,20-dione, and 18,21-dihydroxy-4-pregnene-3,20-dione, but tumor cells produced lesser amounts of dehydroepiandrosterone (DHA), androstenedione, testosterone, and progesterone. Normal cells exposed to ACTH showed an increase in all steroids measured, whereas ACTH-exposed tumor cells showed an increase principally in DHA consistent with a deficiency in 3 beta-hydroxysteroid dehydrogenase activity. Circulating levels of DHA, androstenedione, and testosterone were elevated in the patient before removal of the adrenal tumor. The production of androgens by tumor cells in vitro resembled the pattern of circulating steroids in vivo. These studies demonstrate that tissue culture of human adrenal cells provides a means both to determine their biochemical characteristics and to investigate their responses to exogenous hormones.