Although prolactin (PRL) actions and expression in the brain have been shown, dynamic changes in its intracerebral release and gene expression have still not been demonstrated. Using push-pull perfusion, the in vivo release of PRL was monitored within the paraventricular nucleus (PVN) and medial preoptic area (MPOA) of virgin female, lactating and male rats in response to various stimuli. Perfusion with a depolarizing medium (56 mm K+) increased local release of PRL within both the PVN (P < 0.05) and MPOA (P < 0.05) of urethane-anaesthetized rats, indicating release from excitable neuronal structures. The PRL in perfusates was verified by radioimmunoassay, Nb2 cell bioassays and western blot. Systemic osmotic stimulation (3 m NaCl i.p., 8 mL/kg b.w.) raised PRL concentration in plasma (P < 0.01) but not within the PVN, suggesting independent release from the pituitary and in distinct brain regions. Immobilization for 30 min increased PRL release within the PVN (P < 0.05) and the MPOA (P < 0.01) of virgin female and male (P < 0.05 each) rats and increased hypothalamic PRL mRNA expression (P = 0.008) after 30 and 90 min as revealed by real-time polymerase chain reaction. This indicates a stress-induced activation of both PRL release from and synthesis in hypothalamic neurons. Additionally, PRL was significantly released within, but not outside, the PVN (P < 0.01) and the MPOA (P < 0.05) of lactating rats during suckling and this was accompanied by a significant increase of PRL mRNA (P < 0.05) in the hypothalamus 60 min after suckling. This is the first demonstration of stimulus-induced, locally restricted release and gene upregulation of PRL within the brain, emphasizing the involvement of this ‘novel’ neuropeptide in various brain functions.