The objective of this study was to determine the requirements for the functional luteinization of porcine thecal cells in vitro. In serum-free incubations with luteinizing hormone (LH) (250 ng/ml) androstenedione concentrations increased up to 14 h, after which time no further accumulation occurred; progesterone accumulation was low, and did not increase after 4 h. In the presence of 1% fetal bovine serum and LH, androstenedione production declined, but progesterone production per day increased over a 4-day period, while cellular protein remained constant. LH was required for both the induction and maintenance of elevated progesterone production. Insulin (maximal response at 500 ng/ml) in the presence of 1% serum enhanced the response to LH, causing a dramatic increase in progesterone production, an effect which became greater with time in culture. Dose-response curves for insulin and insulin-like growth factor I (IGF-I) were parallel, but IGF-I was approximately 23-fold more potent than insulin, suggesting that insulin was acting through IGF-I receptors. Our results show that porcine thecal cells, in the presence of LH, insulin or IGF-I, and 1% serum, undergo functional luteinization in vitro, such that androstenedione production declines, and the rate of progesterone production increases with time in culture.