Relaxin 3/INSL 7 has recently been identified as a new member of the insulin/relaxin superfamily. Although it was reported to be dominantly expressed in the brain, its detailed distribution and function in the central nervous system are still obscure. In the present study we demonstrated that in the rat relaxin 3 was mainly expressed in neurons of the nucleus incertus (NI) of the median dorsal tegmental pons. Other relaxin 3-expressing neurons were scattered in the pontine raphe nucleus, the periaqueductal gray and dorsal area to the substantia nigra in the midbrain reticular formation. Relaxin 3-immunoreactive fibers projected particularly densely in the septum, hippocampus, lateral hypothalamus and intergeniculate leaflet of the thalamus. Ultrastructural examination revealed that relaxin 3 was localized in the dense-cored vesicles in the perikarya and was also observed in the synaptic terminals of axons. As almost all relaxin 3-containing neurons express corticotropin-releasing factor (CRF) type 1 receptor in the NI, we examined the response of relaxin 3 neurons to intracerebroventricular administration of CRF; 65% of relaxin 3 neurons expressed c-Fos 2 h after intracerebroventricular administration of 1 microg CRF. We then confirmed that c-Fos was induced in 60% of relaxin 3 neurons in the NI and the expression of relaxin 3 mRNA increased significantly in the NI after water-restraint stress. Collectively, these results suggest that relaxin 3 produced in the NI is released from nerve endings and is involved in the regulation of the stress response.