We have previously found that lactotrophs express and secrete endothelin-like peptides that influence prolactin (PRL) secretion in an autocrine fashion. We have also observed that the incidence of endothelin-immunoreactive lactotrophs is markedly affected by ovarian steroids. In this study, we examined how the ovarian steroid background determines the efficiency of the endothelin-mediated autocrine feedback regulation of PRL secretion. Ovariectomized adult female rats were used throughout these studies. Steroid replacements were made by sc implantation of Silastic capsules immediately following ovariectomy. Eight to 10 wk later, three animals from each treatment group (no steroid control, estradiol, progesterone, estradiol plus progesterone) were sacrificed by decapitation, and the anterior pituitary cells were enzymatically dispersed using collagenase and hyaluronidase. A PRL-specific reverse hemolytic plaque assay was used to measure PRL secretion at the single-cell level. BQ123, a synthetic cyclic pentapeptide with distinctive endothelin-A receptor antagonist quality, caused only a modest elevation of PRL secretion in the control group. Endothelin antagonism did not affect PRL secretion in cells obtained from progesterone-implanted animals. Endothelin antagonism did, however, increase overall PRL secretion in the estradiol and estradiol plus progesterone groups by five- and threefold, respectively. Frequency distribution of PRL plaques in these same two BQ123-treated groups revealed two subpopulations, indicating that lactotrophs differ in their response to endogenous endothelin feedback and that this difference is steroid dependent. These observations clearly suggest that the ovarian steroid milieu (estrogens in particular) can have a profound influence on the self-regulatory mechanisms of lactotrophs. Our results also emphasize that endogenous endothelins may play an important role in the negative feedback regulation of PRL secretion in female rats.