To test the interaction of various hormones on the myometrium, the following experiments were done studying in vitro contractile activity of uterine segments from immature rats. The rats were divided into two groups: group 1 animals were treated with estrogen (n = 9) and group 2 animals were treated with both estrogen and progesterone (n = 11). Uteri from animals in each group were removed and segments were maintained in a temperature- and pH-controlled organ bath. After baseline contractions were established, uterine segments were treated with either oxytocin and then relaxin, or relaxin and then oxytocin. The dose of relaxin used, 20 ng/ml, was previously shown to be effective in inhibiting uterine contractions of animals treated with either estrogen or estrogen plus progesterone. The dose of oxytocin, 2.5 mlU/ml, was the maximal effective dose shown not to produce prolonged tetany. Estrogen plus progesterone treatment increased the frequency of contractions and resulted in contractions of greater duration of the maximal contractile force, as compared with treatment with estrogen alone. Oxytocin caused a stimulation of contractions in relaxin-inhibited uterine strips. Relaxin decreased the hypertonic contractions produced by oxytocin treatment, resulting in contractions similar to baseline. These data demonstrate that oxytocin and relaxin are directly antagonistic in their effects on uterine contractility. This suggests that labor may occur as a result of increased sensitivity to oxytocin or a decreased sensitivity to relaxin.