The myometrium undergoes dramatic changes as pregnancy progresses through phases of proliferation, hypertrophy, contractile state and labor. In this study, we showed that the composition of the muscle actin isoforms, a major component of the myometrial contractile apparatus and cytoskeleton, was modified during pregnancy. The expression of smooth muscle alpha-actin (Acta2, which we abbreviate as alpha-SM-actin) and gamma-actin mRNAs and proteins was examined by real-time polymerase chain reaction and Western immunoblot, and was localized with immunohistochemistry, in the nonpregnant, pregnant, and postpartum rat uterus. Both alpha-SM-actin (vascular specific actin isoform) and gamma-actin (predominant in visceral smooth muscle) were detected in the rat myometrium. Myometrial expression of alpha-SM-actin mRNA and protein was high throughout pregnancy. The transcript and protein levels of gamma-actin were increased significantly in the second part of gestation (31.8-fold increase for mRNA and 16.7-fold increase for protein relative to nonpregnant). The localization of gamma-actin was markedly altered during pregnancy. In early gestation, myometria from empty and gravid uterine horns of the unilaterally pregnant rats showed abundant gamma-actin immunostaining in the longitudinal layer but weak staining in the circular layer. Gamma-actin immunostaining increased in only the circular layer of the gravid horn after midgestation and remained low in the empty one. Gamma-actin protein translocated to the membranous region of uterine myocytes at late gestation. The temporal alteration in gamma-actin expression and localization at late gestation suggested that this change in myometrial composition of contractile proteins is important to adequately prepare the myometrium for the development of optimal contractions during labor.