Chronic cytomegalovirus (CMV) infections are implicated in vascular diseases. Recently, we showed that an active mouse CMV (mCMV) infection in nonpregnant mice increased endothelial-dependent vasodilation in isolated mesenteric and uterine arteries. In late pregnancy, while increased vasodilation was found in mesenteric arteries from infected mice, there was a dramatic decrease in uterine arteries. Understanding the mechanisms for these vascular changes during CMV infections is important for pregnancy outcomes and long-term consequences of this chronic infection. Increased nitric oxide (NO) is implicated in CMV-associated atherosclerosis, and CMV replication is dependent on prostaglandin H synthase (PGHS) activity. Alternatively, CMV infections decrease NO under inflammatory conditions. We therefore hypothesized that changes in the contribution by NO or PGHS-induced vasodilators would explain the increased or decreased endothelial-dependent vasodilation in arteries from nonpregnant and late pregnant mice, respectively. We found that the contribution by NO to methacholine-induced vasodilation was significantly increased in mesenteric, but not uterine, arteries isolated from nonpregnant and pregnant mCMV-infected mice. Prostaglandin inhibition did not affect endothelial-dependent vasodilation in any group. Vasodilation responses to sodium nitroprusside, an NO donor, were increased in mesenteric and uterine arteries isolated only from mCMV-infected nonpregnant mice. These results explain the increased vasodilation responses observed in mesenteric arteries from mCMV-infected mice; however, the decreased vasodilation in uterine arteries from pregnant mice could not be explained by these mechanisms. Thus CMV infection affects the contribution of NO differently in endothelial-dependent vasodilation in pregnant compared with nonpregnant mice and also in the mesenteric compared with the uterine vascular bed.