The balance between relaxin and prolactin has been suggested to be significant in the control of uterine activity during pregnancy. The current study was designed to determine the effect of rat prolactin on rat uterine horn segment contractility and its interaction with relaxin. Rat uterine segments mounted on a smooth muscle transducer were treated sequentially with prolactin and relaxin, and stimulated electrically. No stimulatory effect of prolactin was noted. Relaxin-inhibited uterine horn segment activity was not altered by prolactin. The reported stimulatory effect of prolactin may have been a pharmacologic effect of human prolactin on rat uterine horn segments, or may have resulted from contaminants in the preparations used.