Prolactin signalling within hypothalamic areas associated with the control of fertility was examined in male and lactating female rats. Following exogenous prolactin treatment, phosphorylation of STAT5 (signal transducer and activator of transcription) within the arcuate nucleus was measured using a highly sensitive immunoblotting strategy. A significant increase in phosphorylated STAT5 was detected in the arcuate nucleus of female rats compared with same-sex controls. No such effect was apparent in the males. Phosphorylation of STAT5 was not observed in the liver of either males or females. These results show that prolactin-induced intracellular signalling within the hypothalamus involves activation of the Janus tyrosine kinase/signal transducer and activator of transcription pathway, and that this signalling mechanism can be readily triggered in lactating females where prolactin receptors are known to be upregulated and fertility impaired.