Relaxin-3 is a newly discovered member of the insulin superfamily. It is expressed in the nucleus incertus (NI) of the brainstem which has projections to the hypothalamus. Relaxin-3 binds with high affinity to the G-protein-coupled receptors RXFP1 and RXFP3. RXFP3 is expressed predominantly in the CNS, and in particular within the hypothalamic paraventricular nucleus (PVN). The physiological function of relaxin-3 is unknown but recent work suggests it may play a role in appetite control. Most relaxin-3 immunoreactive neurons in the NI co-express the corticotrophin releasing factor (CRF) type 1 receptor. Central administration of CRF results in expression of the early gene c-fos in 65% of relaxin-3 neurons in the NI and similar c-fos expression is achieved under conditions of stress. These findings, together with the presence of relaxin-3 and its receptors in the PVN led us to investigate the effect of central administration of relaxin-3 on the hypothalamo-pituitary-adrenal axis (HPG) in male rodents. Intraparaventricular (iPVN) administration of relaxin-3 (540 pmol) significantly increased ACTH at 15 and 30 minutes following injection compared to vehicle [51.4±4.8 pg/ml (vehicle) vs 114.8±18.0 pg/ml (relaxin-3) at 15 min, P< 0.05 vs vehicle and 61.4±10.4 pg/ml (vehicle) vs 104.5±14.4 pg/ml (relaxin-3) at 30 min, P< 0.05 vs vehicle]. This was associated with a rise in corticosterone at 15 and 30 minutes in the relaxin-3 treated group compared to vehicle [252.8±46.8 ng/ml (vehicle) vs 424.3±47.3 ng/ml (relaxin-3) at 15 min, P< 0.05 vs vehicle and 122.0±32.2 ng/ml (vehicle) vs 497.6±60.3 ng/ml (relaxin-3) at 30 min, P<0.001 vs vehicle]. The stress hormone prolactin was also significantly raised following intraPVN administration of relaxin-3 (540 pmol) at 15 and 30 minutes following injection compared to vehicle. These results suggest that relaxin-3 may play an important role in the control of stress and the hypothalamo-pituitary-adrenal axis.