The production of inhibin by isolated segments of seminiferous tubules from adult male rats cultured in vitro was investigated using a heterologous specific radioimmunoassay. Increasing lengths of tubules (5,10, 20 and 40 cm) maintained in culture for 4 or 5 days produced increasing amounts of inhibin in vitro. A dose-dependent increase in inhibin production was observed after stimulation with ovine follicle-stimulating hormone (FSH)-s17 (0.1-1000 ng/ml). The tubule segments remain sensitive to FSH stimulation for up to 20 days of culture despite a progressive decline in basal inhibin production, resulting in an increase in the magnitude of the response to FSH stimulation between 0-5, 5-10 and 10-20 days of culture. In the presence of the protein synthesis inhibitor, cycloheximide (50 μg/ml), both basal and FSH-stimulated inhibin secretion are inhibited. Testosterone (10-8-10-5 M) does not affect basal inhibin production, although inhibition of the FSH-induced production of inhibin occurred at only the highest dose of testosterone used (10-5 M). These data demonstrate that the production of inhibin by segments of seminiferous tubules from adult male rats can be used to study the control of inhibin secretion. © 1988.