Placental ischemia during pregnancy is thought to release cytokines such as tumor necrosis factor-α (TNF-α), which may contribute to the increased vascular resistance associated with pregnancy-induced hypertension. We have reported that a chronic twofold elevation in plasma TNF-α increases blood pressure in pregnant but not in virgin rats; however, the vascular mechanisms are unclear. We tested the hypothesis that increasing plasma TNF-α during pregnancy impairs endothelium-dependent vascular relaxation and enhances vascular reactivity. Active stress was measured in aortic strips of virgin and late-pregnant Sprague-Dawley rats untreated or infused with TNF-α (200 ng · kg⁻¹ · day⁻¹ for 5 days) to increase plasma level twofold. Phenylephrine (Phe) increased active stress to a maximum of 4.2 ± 0.4 × 10³and 9.9 ± 0.7 × 10³ N/m² in control pregnant and TNF-α-infused pregnant rats, respectively. Removal of the endothelium enhanced Phe-induced stress in control but not in TNF-α-infused pregnant rats. In endothelium-intact strips, ACh caused greater relaxation of Phe contraction in control than in TNF-α-infused pregnant rats. Basal and ACh-induced nitrite/nitrate production was less in TNF-α-infused than in control pregnant rats. Pretreatment of vascular strips with 100 μM N ^G-nitro-l-arginine methyl ester, to inhibit nitric oxide (NO) synthase, or 1 μM 1 H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one, to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in control but not in TNF-α-infused pregnant rats. Phe contraction and ACh relaxation were not significantly different between control and TNF-α-infused virgin rats. Thus an endothelium-dependent NO-cGMP-mediated vascular relaxation pathway is inhibited in late-pregnant rats infused with TNF-α. The results support a role for TNF-α as one possible mediator of the increased vascular resistance associated with pregnancy-induced hypertension.