Normal pregnancy is characterized by an increased uterine blood flow due to growth and remodeling of the maternal uterine vasculature and enhanced vasodilation of the uterine arteries. The objective of the present study was to examine the role of endothelial cell Ca²⁺ signaling in augmented endothelium-mediated vasodilation of uteroplacental arteries in late pregnancy. We performed fura 2-based measurements of the concentration of Ca²⁺ in the cytoplasm [Ca²⁺]_iof endothelial cells simultaneously with diameter in pressurized uterine arteries from non-pregnant (NP) and late pregnant (LP) rats. Basal levels of endothelial cell [Ca²⁺]_i were higher in arteries from LP rats compared to NP controls. Withdrawal of extracellular Ca²⁺ resulted in a decrease in the level of basal [Ca²⁺]_i that was significantly larger in arteries of LP than NP rats. The rate of Mn²⁺-induced quenching in fura 2 fluorescence was significantly elevated in late pregnancy implicating augmented Ca²⁺ influx as a cause of increased basal levels of [Ca²⁺]_i in endothelial cells. Elevation of intraluminal pressure resulted in a transient increase in endothelial [Ca²⁺]_i that was markedly potentiated in late gestation. ACh-induced [Ca²⁺]_i and vasodilator responses were significantly augmented in arteries of LP compared to NP rats and were abolished by BAPTA treatment, demonstrating a critical role of [Ca²⁺]_i elevation in the production of endothelium-derived vasodilators. Together, these results indicate that late pregnancy is a state of enhanced basal and stimulated Ca²⁺ signaling in endothelial cells of uterine vessels, which may represent an important underlying mechanism for augmented vasodilation in the maternal uterine circulation.