Objective: The purpose of this study was to evaluate the effects of recombinant human relaxin on the uterine artery and myometrial contractility in pregnant rats. Study Design: Uterine artery and myometrial rings from mid and term pregnant rats were used. Relaxin effect was studied on phenylephrine-induced contraction in the presence or absence of nitric oxide synthase inhibitor, Nω-nitro-l-arginine methyl ester, soluble guanylate cyclase inhibitor, 1H-oxadiazolo-quinoxaline-1-one, or adenylate cyclase inhibitor, SQ-22,536. The myometrial inhibitory effect of relaxin was studied on spontaneous and oxytocin- or protein kinase C activator-induced contractions. Results: Uterine artery relaxation by relaxin was greater at mid pregnancy compared with term. Relaxin effect was decreased by SQ-22,536, 1H-oxadiazolo-quinoxaline-1-one and Nω-nitro-l-arginine methyl ester at mid pregnancy. Relaxin inhibited spontaneous contractions at mid pregnancy but not at term. Relaxin had no effect on oxytocin- or indolactam-V-induced contractions. Conclusion: Relaxin effect is mediated by nitric oxide, soluble guanylate cyclase, and adenylate cyclase in mid pregnant uterine artery. Relaxin inhibits spontaneous uterine activity at mid pregnancy. Relaxin effect decreased at term gestation in both tissues. (Am J Obstet Gynecol 2003;188:1468-76.)