Hyperprolactinaemia causes infertility, but the mechanisms involved are not known. The present study aimed to determine if and how prolactin may influence LH secretion in the adult female mouse. Using ovariectomised, estrogen-treated (OVX+E) mice, we found that seven days of icv prolactin potently suppressed serum luteinizing hormone (LH) levels (p<0.05). To examine whether this central action of prolactin may involve the gonadotropin-releasing hormone (GnRH) neurons, the effects of acute and chronic prolactin upon CREB phosphorylation (pCREB) in GnRH neurons were examined using dual-label immunocytochemistry. In diestrous and OVX+E mice, a single sc injection of ovine prolactin resulted in a significant (p<0.05) doubling of the number of GnRH neurons expressing pCREB. OVX+E mice treated with 5 injections of ovine prolactin over 48h showed a 4-fold increase in the number of GnRH neurons with pCREB. To determine whether GnRH neurons might be regulated directly by prolactin, we examined prolactin receptor (PRL-R) mRNA expression in green fluorescent protein (GFP)-tagged GnRH neurons by single cell RT-PCR. As a positive control, PRL-R mRNA was measured in arcuate dopaminergic neurons obtained from GFP-tagged tyrosine hydroxylase neurons. Three of 23 (13%) GnRH neurons were identified to express PRL-R transcripts whereas 9 or 11 (82%) of arcuate dopaminergic neurons were found to co-express PRL-R mRNA. These data demonstrate that prolactin suppresses LH levels in the mouse, as it does in other species, and indicate that it acts centrally to regulate intracellular signaling within GnRH neurons. This is likely to occur, at least in part, through the direct regulation of a sub-population of GnRH neurons.