Recently, we demonstrated that relaxin (RLX), a peptide hormone of ovarian origin, inhibits growth and promotes differentiation of MCF-7 breast adenocarcinoma cells. We also showed that RLX stimulates the production of nitric oxide (NO) in several cell types. NO has been reported to have antitumor activity by inhibiting proliferation, promoting differentiation, and reducing the metastatic spread of some tumor cell types. In this study, we aimed at evaluating whether RLX influences the L-arginine-NO pathway in MCF-7 cells. The cells were grown in the absence or presence of RLX at different concentrations, and cell proliferation, constitutive and inducible NO synthase activities, nitrite production, and intracellular levels of cyclic GMP were investigated. The results obtained indicate that RLX increases inducible NO synthase activity and potentiates NO production. This was accompanied by an elevation of intracellular cyclic GMP, which is known to mediate the cell response to NO. The RLX-induced activation of the L-arginine-NO pathway in the MCF-7 cells was inversely related to the rate of cell proliferation. These results suggest that the cytostatic effect of RLX on MCF-7 breast cancer cells may rely on its ability to stimulate endogenous production of NO.