We have previously found that the ovarian steroid background determines the efficiency of the endothelin-mediated autocrine feedback regulation of prolactin (PRL) secretion. In this study, we investigated the role of endogenous endothelins in regulating PRL secretion during the estrous cycle. Adult female rats representing different stages of the 4-d cycle were sacrificed by decapitation, and the anterior pituitary cells were enzymatically dispersed using collagenase and hyaluronidase. PRL secretion of individual lactotrophs was measured in a PRL-specific reverse hemolytic plaque assay, and the influence of endogenous endothelins on PRL secretion was assessed by applying the selective ET(A) receptor antagonist peptide, BQ123. Blocking the endothelin-mediated autocrine feedback resulted in an increase in PRL secretion when cells were obtained at proestrus, estrus, and diestrus-1, whereas PRL secretion was decreased at diestrus-2 by ET(A) receptor blockade. These observations suggest that endogenous endothelins are predominantly inhibitory during proestrus, estrus, and diestrus-1, whereas at diestrus-2 their influence on PRL secretion is stimulatory. Whereas the bell-shaped concentration-response curves with BQ123 at proestrus and diestrus-1 may indicate a transition state in which endogenous endothelins can be both stimulatory and inhibitory, at estrus the influence of endogenous endothelins is unequivocally inhibitory in nature. We propose that intensification of the endogenous endothelin- mediated negative feedback at estrus may play a role in restraining PRL secretion following the estradiol- induced proestrous PRL surge.