The insulin superfamily, characterized by common disulphide bonds, includes not only insulin but also insulin-like peptides such as relaxin-1 and relaxin-3. The actions of relaxin-3 are largely unknown but recent work suggests a role in regulation of food intake. Relaxin-3 mRNA is highly expressed in the nucleus incertus which has extensive projections to the hypothalamus and relaxin immunoreactivity is present in several hypothalamic nuclei. In the rat, relaxin-3 binds and activates both RXFP1, which also binds relaxin-1, and a previously orphaned G-protein coupled receptor, RXFP3. These receptors are extensively expressed in the hypothalamus. The aims of these studies were two fold: firstly to map the hypothalamic site(s) of the orexigenic action of relaxin-3 and secondly to examine the site(s) of neuronal activation following central relaxin-3 administration. After microinjection into hypothalamic sites, human relaxin-3 (H3) (180 pmol) significantly stimulated 0-1 h food intake in the supraoptic nucleus (SON), arcuate nucleus (ARC) and the anterior preoptic area (APOA) [SON 0.4 ±0.2g (vehicle) vs 2.9 ± 0.5g (H3), p<0.001, ARC 0.7 ± 0.3g (vehicle) vs 2.7 ±0.2g (H3), p<0.05 and APOA 0.8 ± 0.1g (vehicle) vs 2.2 ±0.2g (H3), p<0.05]. Cumulative food intake was significantly increased up to 8 hours following administration into the SON and 4 hours into the APOA. A significant increase in fos-like immunoreactivity (FLI) was seen in the SON following central relaxin-3 administration. Relaxin-3 stimulates feeding in several hypothalamic nuclei and these studies provide further support for relaxin-3 as an important peptide in appetite regulation.