Magnocellular neurons of the supraoptic (SON) and paraventricular (PVN) nuclei show considerable plasticity during pregnancy and lactation. Prolactin receptors (PRL-R) have been identified in both these nuclei. The aim of this study was to investigate the cell type(s) expressing mRNA for the long form of prolactin receptor (PRL-R_L), and to determine whether patterns of expression change during pregnancy and lactation. In addition, we examined effects of prolactin on excitability of oxytocin and vasopressin neurons. Sections from brains of non-pregnant, pregnant and lactating rats were hybridized with an ³⁵S-labelled probe to label PRL-R_L mRNA together with digoxigenin-labeled probes to detect either oxytocin or vasopressin mRNA. In the SON, PRL-R_L mRNA was predominantly colocalised with oxytocin mRNA, with over 80% of oxytocin neurons positive for PRL-R_L mRNA. Very few (<10%) vasopressin neurons expressed PRL-R_L mRNA. In the PVN, PRL-R_L mRNA was also predominantly found in oxytocin neurons, and the proportion of PRL-R_L-positive oxytocin neurons increased significantly during pregnancy and lactation. As in the SON, relatively few vasopressin cells contained PRL-R_L mRNA. For in vivo electrophysiology, non-pregnant rats were anesthetized, then extracellular single neuron activity recorded in identified oxytocin and vasopressin neurons. Following a period of baseline recording, the effect of prolactin (1 µg i.c.v.) on firing rate was examined. Prolactin treatment of non-pregnant rats induced a significant decrease in firing rates of oxytocin neurons. There was no effect of prolactin on the activity of vasopressin neurons. Together, these data provide strong evidence that prolactin directly and specifically regulates activity of oxytocin neurons.