To determine whether gestation-related changes in responsiveness of the rat uterus to β-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12–14) and then late (Days 18–20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the β-adrenergic receptor (isoproterenol, 10⁻⁴ M), the G protein (GTP, 10⁻⁵ M), or the adenylyl cyclase enzyme (MnCl₂, 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [³H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor.