Synthesis and secretion of testosterone by testes of guinea pig fetuses were studied in organ culture. The amount of testosterone secreted into the culture medium was estimated by radio-immunoassay. It was demonstrated that testosterone was already secreted by testes explanted at 25 days. The amount of testosterone secreted during the first day in culture by testes from fetuses of different ages (25-30 days) increased with fetal age. The amount of testosterone extracted from fetal guinea pig testes at ages used for explantation (25-30 days) were much lower than the amount secreted during the first 24 hours in culture. During subsequent days in culture, an increase in the amount of testosterone secreted was observed only for testes explanted at 25 days. The amount secreted by testes from older fetuses (26-30 days) stayed constant or decreased. Aminogluthetimide phosphate (100 micron) decreased significantly testosterone secretion by testes explanted at 25 days. These findings indicate that the capacity for biosynthesis of testosterone is present in fetal guinea pig testes at 25 days and increases during the subsequent days.