The effects of androgen or glucocorticoid on the induction of secretory proteins in SC-3 cells (a cloned cell line from Shionogi carcinoma 115) were examined in a serum-free medium [Ham's F-12: Eagle's minimum essential medium (1:1, v/v) containing 0.1% bovine serum albumin]. Through analysis of [35S]methionine-labeled proteins by one-dimensional (sodium dodecyl sulfate polyacrylamide) gel electrophoresis, we successfully demonstrated the production of a testosterone (10(-9)-10(-6) M)- or dexamethasone (10(-8) M)-induced secretory protein with a molecular weight of 24,000 in SC-3 cells. However, 17 beta-estradiol (10(-8) M) or progesterone (10(-8) M) had no such effect. The production of the secretory 24K protein increased slightly within 2 hr after the addition of 10(-8) M testosterone and increased further thereafter. The addition of tunicamycin to the incubation mixture with [35S]methionine resulted in the identification of a testosterone- or dexamethasone-induced secretory protein with a molecular weight of 20,000. By the use of two-dimensional gel electrophoresis, we could demonstrate that both testosterone (10(-8) M) and dexamethasone (10(-8) M) added to the serum-free medium stimulate the secretion from SC-3 cells of the same 5 proteins (30-40K, 39K, 30K, 27K proteins and the secretory 24K glycoprotein). The present findings show that physiological concentrations of both androgen and glucocorticoid induce the secretion of the same 5 proteins from SC-3 cells.