Levels of LH and FSH released in vitro by rat anterior pituitary cells which were either co-cultured with isolated rat Sertoli cells or were grown with spent media recovered from the cultured Sertoli cells were measured by radioimmunoassay. The amounts of FSH released by pituitary cells grown for three days with Sertoli cells isolated from 31-36 day old rats or spent media from the cultured Sertoli cells, were significantly (P less than 0.01) lower compared to control pituitary cultures grown with fresh chemically defined medium. In contrast, the levels of LH were similar to the controls. The selective inhibition of FSH release was not observed when pituitary cells were co-cultured with rat spleen or kidney cells or with ruptured Sertoli cells. The FSH-inhibiting Sertoli-cell factor (SCF) was found to be a heat-labile macromolecule. It is suggested that the SCF may be secreted by the Sertoli cells in vivo, and regulate FSH secretion via negative feedback mechanism at the pituitary level.