17 beta-Estradiol (E2) was found to affect gonadotrophs in long term ovine pituitary cell culture in several ways which were easily measured. It inhibited basal FSH secretion and, concomitantly, stimulated LH secretion. High physiological levels of E2 (10(-10) M; 27 pg/ml) were able to elicit about 70% of the maximal responses, and the effects were developed fully between 6-24 h after the addition of E2. In culture, the antiestrogens (AEs) tamoxifen, nafoxidine, and CI-680 antagonized both the inhibitory and stimulatory effects of E2 (10(-10) M) on gonadotropin secretion in a dose-dependent manner, and 10(-6) M AE was fully effective. At concentrations of 10(-6) M or lower, the AEs showed no significant intrinsic estrogenicity. At higher concentrations, the AEs progressively altered gonadotropin secretion in an apparently nonspecific fashion. Preincubations of cultures with AEs (3.3 X 10(-6) M) antagonized E2 action (10(-10) M) for approximately 18-24 h after the removal of AEs. Tamoxifen appeared to be the most potent AE tested. In conclusion, long term ovine pituitary cell cultures have provided a useful in vitro system for testing the biological potencies of AE analogs. AEs appeared to act as pure estrogen antagonists in ovine gonadotrophs.