Relaxin-3 (RLN3) and its native receptor, relaxin family peptide 3 receptor (RXFP3), constitute a newly identified neuropeptide system enriched in mammalian brain. The distribution of RLN3/RXFP3 networks in rat brain and recent experimental studies suggest a role for this system in modulation of arousal, stress, metabolism, and cognition. In order to facilitate exploration of the biology of RLN3/RXFP3 in complementary murine models, this study mapped the neuroanatomical distribution of the RLN3/RXFP3 system in mouse brain. Adult, male wildtype and RLN3 knock-out (KO)/LacZ knock-in (KI) mice were used to map the central distribution of RLN3 gene expression and RLN3-like immunoreactivity (-LI). The distribution of RXFP3 mRNA and protein was determined using [(35)S]-oligonucleotide probes and a radiolabeled RXFP3-selective agonist ([(125)I]-R3/I5), respectively. High densities of neurons expressing RLN3 mRNA, RLN3-associated beta-galactosidase activity and RLN3-LI were detected in the nucleus incertus (or nucleus O), while smaller populations of positive neurons were observed in the pontine raphé, the periaqueductal gray and a region adjacent to the lateral substantia nigra. RLN3-LI was observed in nerve fibers/terminals in nucleus incertus and broadly throughout the pons, midbrain, hypothalamus, thalamus, septum, hippocampus, and neocortex, but was absent in RLN3 KO/LacZ KI mice. This RLN3 neural network overlapped the regional distribution of RXFP3 mRNA and [(125)I]-R3/I5 binding sites in wildtype and RLN3 KO/LacZ KI mice. These findings provide further evidence for the conserved nature of RLN3/RXFP3 systems in mammalian brain and the ability of RLN3/RXFP3 signaling to modulate "behavioral state" and an array of circuits involved in arousal, stress responses, affective state, and cognition.