Mouse testes were cultured for 19–20 days at either 31 or 37 °C with a change of medium every 4 days. After treatment with charcoal and dextran T, the recovered testis media were incubated with rat anterior pituitary cells, and secretions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were estimated by radioimmunoassay 3 days later. FSH release was significantly lowered when pituitary cells were grown with media of testes cultured at 31 °C compared to cultures grown with fresh medium or with media of testes cultured at 37 °C for more than 4 days. LH secretion was normal in one experiment and reduced in the other with the media of testes cultured at 31 °C. Treatment of testicular media by heat or trypsin reduced the inhibiting activity. After 8 days at 37 °C, both germinal and Sertoli cells were damaged in the testis cultures, while at 31 °C germinal cells alone were destroyed, Sertoli cells remained normal. These studies suggest that (1) a substance which responds to the definition of inhibin (protein – preferentially acting on FSH) is secreted in the medium of testis culture; (2) inhibin is produced by Sertoli cells; (3) inhibin is secreted only if the temperature is inferior to 37 °C.