In vitro steroid production by a virilizing Sertoli-Leydig cell tumor of the ovary was studied. For comparison, stromal tissue from the opposite normal ovary was also incubated under similar conditions. The tumor fragments secreted significantly more testosterone (527 +/- 168 versus 48 +/- 29 pg/mg tissue, p less than 0.001), androstenedione (1188 +/- 400 versus 40 +/- 10 pg/mg tissue, p less than 0.001), and dehydroepiandrosterone (419 +/- 132 versus 73 +/- 25 pg/mg tissue, p less than 0.004) than that of normal ovarian stroma. Measurement of steroids in the ovarian venous serum draining the tumor indicated a peripheral ovarian gradient for both delta 4 and delta 5 steroids. Incubation of tumor fragments with luteinizing hormone alone resulted in a significant increase in the secretion of androstenedione and dehydroepiandrosterone (p less than 0.05). Addition of insulin to luteinizing hormone resulted in significantly greater release of androstenedione than that of treatment with luteinizing hormone alone (p less than 0.04). Addition of insulin had no effect on the release of dehydroepiandrosterone. Luteinizing hormone and insulin, either alone or in combination, failed to produce any change in the secretion of testosterone. We conclude that (1) increased testosterone secretion by Sertoli-Leydig cell tumor resulted from increased availability of precursors from both delta 4 and delta 5 pathways; (2) the tumor was responsive to luteinizing hormone with an increase in the secretion of androstenedione and dehydroepiandrosterone; (3) insulin acts synergistically with luteinizing hormone to increase secretion of androstenedione; (4) the tumor has specific binding sites for insulin; and (5) the increased levels of insulin and luteinizing hormone in polycystic ovarian disease may play a role in the pathogenesis of Sertoli-Leydig cell tumor.