The secretion of follicle-stimulating hormone (FSH) by the pituitary gland appears to be partially regulated by a protein hormone, inhibin, which is produced in the Sertoli cells of the testis and is also present in ovarian follicular fluid (FF). The aim of the present study was to develop a sensitive method for the detection and estimation of inhibin-like activity, using dispersed pituitary cells in culture. Pituitary cells from adult male rats were dispersed and precultured for 3 days. After renewal of the medium (2 ml), samples to be tested for inhibin-like activity were added and culture was continued for a further 3 days. A dose-dependent suppression of the concentration of FSH in the medium (λ = 0·17–0·22) was observed after addition of FF (0·05–1 μl) or Sertoli cell culture medium (SCCM, 0·05–1 ml). Luteinizing hormone (LH) concentrations were not affected with these doses of FF, but SCCM and higher doses of FF caused a significant increase in the concentration of LH in the medium. During an additional 6 h of culture in the presence of luteinizing hormone releasing hormone (LH-RH), FF and SCCM suppressed the release of both FSH (λ = 0·07–0·11) and LH in a dose-dependent way. Cellular content of FSH, but not of LH, was decreased after these treatments. These results could not be explained by damage to the pituitary cells, by degradation of FSH or LH-RH, or by effects of steroids. It is concluded that this pituitary cell culture system can be used as a sensitive method for the estimation of inhibin-like activity in FF and SCCM.