Previously, we demonstrated that endogenous circulating relaxin promotes growth of the mammary nipples during the second half of pregnancy in the rat. The objective of this study was to determine whether relaxin acts directly on rat nipples to promote their growth. Initially, specific relaxin-binding cells were identified to assure that relaxin binds to the same cell types in the nipples of nonpregnant rats as those we previously described in pregnant rats. To examine relaxin-induced growth of the mammary nipples, 5 days after ovariectomy, 48 nonpregnant rats were assigned (12 rats/group) to 1 of the following 4 treatment groups: ovariectomized controls, estrogen treated, relaxin treated, and estrogen plus relaxin treated. Estrogen (0.05 micrograms 17 beta-estradiol) or estradiol vehicle (0.1 ml stripped corn oil) was administered sc on the dorsal side of the neck daily for the entire 10-day treatment period. Porcine relaxin (12.5 micrograms) or relaxin vehicle (0.05 ml 5% beeswax in corn oil) was administered sc at the base of the left abdominal nipple daily for the last 5 days of the 10-day treatment period. After hormone treatments, the lengths and wet weights of the left (relaxin-treated) and right (untreated) abdominal nipples were measured. There were three findings. First, the presence of specific relaxin binding in the epithelial cells of the lactiferous duct, smooth muscle cells, and skin of the nipples in nonpregnant rats was identical to the sites of specific relaxin binding in the nipples of pregnant rats. Second, relaxin-induced increases in nipple length and wet weight were mediated at least in part by the direct effects of relaxin in the nipple. Third, estrogen was not required for relaxin-induced increases in nipple length and wet weight. We conclude that relaxin stimulates the growth of rat mammary nipples at least in part through direct actions in the nipples, and that estrogen is not required for these actions.