Uterine myometrial contractility remains a poorly characterized area of research in reproductive physiology. Rnd1, a novel member of the GTP-binding Rho protein family, inhibits Ca(2+)-sensitization by specifically interfering with a RhoA/Rho-activated kinases-dependent mechanism in smooth muscle. In addition to Rnd1, there are two other members, Rnd2 and Rnd3, in the Rnd family of Rho proteins. In the present comparative study of myometrial contractility in rats and humans, we found that all three Rnd mRNAs were expressed in nonpregnant rat myometrium and in nonpregnant human myometrial tissues. Although all three mRNA levels increased significantly after gestation in rat myometria, only Rnd1 expression was significantly greater after gestation in human samples. In the ovariectomized rat, administration of estrogen and/or progesterone increased the expression of all Rnd mRNAs. These results suggest that universal Rnd family up-regulation during pregnancy in rats may have an important role for negative-feedback control of uterine contraction during gestation by inhibiting RhoA-mediated increase in Ca(2+) sensitivity of contractile elements. Such increases in Rnd levels may be due to augmented levels of reproductive steroids in rats. Our data also point to gestational differences between rats and humans in Rnd isoform patterns.