Hyperprolactinaemia can reduce fertility and libido. Although central prolactin actions are thought to contribute to this, the mechanisms are poorly understood. We first tested whether chronic hyperprolactinaemia inhibited two neuroendocrine parameters necessary for female fertility: pulsatile LH secretion and the estrogen-induced LH surge. Chronic hyperprolactinaemia induced by the dopamine antagonist sulpiride caused a 40% reduction LH pulse frequency in ovarectomized rats, but only in the presence of chronic low levels of estradiol. Sulpiride did not affect the magnitude of a steroid-induced LH surge or the percentage of GnRH neurons activated during the surge. Estradiol is known to influence expression of long form prolactin receptors (PRL-R) and components of prolactin's signaling pathway. To test the hypothesis that estrogen increases PRL-R expression and sensitivity to prolactin, we next demonstrated that estradiol greatly augments prolactin-induced STAT5 activation. Lastly, we measured PRL-R and suppressor of cytokine signaling (SOCS-1, -3 and CIS, which reflect the level of prolactin signaling) mRNAs in response to sulpiride and estradiol. Sulpiride only induced SOCS-1 in the in the medial preoptic area, where GnRH neurons are regulated, but in the arcuate nucleus and choroid plexus PRL-R, SOCS-3 and CIS mRNA levels were also induced. Estradiol enhanced these effects on SOCS-3 and CIS. Interestingly, estradiol also induced PRL-R, SOCS-3 and CIS mRNA levels independently. These data show that GnRH pulse frequency is inhibited by chronic hyperprolactinaemia in a steroid-dependent manner. They also provide evidence for estradiol-dependent and brain region-specific regulation of PRL-R expression and signaling responses by prolactin.