The differential feedback actions of estrogen (E2) on gonadotropin secretion were studied by means of an in vivo isolated pituitary paradigm. Adult female rats were hypophysectomized (hypox) and the next day received single anterior pituitary transplants (graft) under the kidney capsule. At the same time rats underwent bilateral ovariectomy. On the third day each animal was fitted with a catheter system which allowed for intermittent infusions of LHRH (250 ng/5 min.h) and chronic blood sampling. Rats received LHRH infusions for 7 days. On the sixth day of LHRH infusions blood samples were collected for 4 h 5, 15, 25, 35, 45 min after each hourly LHRH pulse. After 1 h of sampling, animals received sc injections of 2 micrograms estradiol benzoate (EB; n = 5) or oil vehicle (n = 5). Plasma LH, FSH, E2, and PRL levels in samples from all groups were determined by RIA. In hypox/graft rats LH release, but not FSH release, was pulsatile in response to the hourly LHRH infusions. Injection of EB in the hypox/graft rats significantly (P less than 0.05) suppressed LH release within 3 h by 57%, while FSH was unaffected. PRL levels were elevated by approximately 10-fold in the hypox/graft animals compared to those in pituitary-intact rats. These levels, however, were not changed as a function of steroid treatment and, therefore, could not account for the effects of EB on LH secretion. On the basis of these observations we conclude that 1) a major inhibitory effect of an acute injection of EB on LH secretion is exerted by a direct action on pituitary gonadotropes, and 2) E2 can differentially affect the release of LH and FSH by an intrapituitary mechanism. It is hoped that development of this model will allow for further investigation of the cellular mechanisms that mediate feedback actions of E2 on pituitary gonadotropes exposed to intermittent LHRH stimulation.