Relaxin is a polypeptide hormone that inhibits rat uterine contractions. To test the hypothesis that the mechanism of action of this effect may involve shifts in calcium ions, the biologic action of relaxin on isolated rat uterine horns was directly correlated with measurements of 45Ca2+ efflux from and uptake into the tissues. Kinetic analysis demonstrated that the efflux of 45Ca2+ from rat uterine horns was significantly faster when tissue was incubated with 25 ng/ml relaxin as compared with control tissues incubated with no relaxin. In addition, at the end of the efflux experiments, control uteri contained 3.9% of the starting 45Ca2+, whereas the relaxin-treated uteri contained only 2.55%, indicating greater total Ca2+ efflux from the relaxin-treated horns (p less than 0.05). The effect of relaxin on 45Ca2+ uptake by uterine tissue was also studied. Analysis of uptake curves by linear regression demonstrated that relaxin treatment leads to less total uptake of 45Ca2+ in the uterine tissue, although the differences are not statistically significant. These experiments demonstrate that relaxin inhibition of rat uterine contractions in vitro is associated with a decrease in intracellular free Ca2+, caused, at least in part, by the promotion of Ca2+ efflux. These results represent the first step in defining the mechanism of action of this hormone.