In pregnancy, the uterine vasculature undergoes dramatic vasodilatory adaptations. Previously, vascular endothelial growth factor (VEGF) has been shown to stimulate endothelial nitric oxide synthase (eNOS) in uterine artery endothelial cells (UAECs) derived from pregnant ewes to a greater extent than those from non-pregnant ewes in a manner not fully explained by changes in the phosphorylation of eNOS. In this study, we used Fura-2 Ca(2+) imaging and arginine-to-citrulline conversion eNOS activity assays to assess the importance of VEGF-stimulated Ca(2+) responses in pregnancy-related changes in NO production in UAEC. In this study, we show that pregnancy-induced changes in VEGF-stimulated Ca(2+) responses could account in part for the greater capacity of VEGF to stimulate eNOS in UAECs from pregnant versus non-pregnant animals. VEGF-stimulated Ca(2+) responses in UAECs from pregnant and non-pregnant animals were mediated through VEGF receptor 2 and were detected in roughly 15% of all cells. There were no pregnancy-specific differences in area under the curve or peak height. UAECs from pregnant animals were more consistent in the time to response initiation, had a larger component of extracellular Ca(2+) entry, and were more sensitive to a submaximal dose of VEGF. In UAECs from pregnant and non-pregnant animals Ca(2+) responses and eNOS activation were sensitive to the phospholipase C/inositol 1,4,5-trisphosphate pathway inhibitors 2-aminoethoxydiphenylborane and U73122. Thus, changes in VEGF-stimulated [Ca(2+)]i are necessary for eNOS activation in UAECs, and pregnancy-induced changes in Ca(2+) responses could also in part explain the pregnancy-specific adaptive increase in eNOS activity in UAECs.