Tumor necrosis factor- (TNF) is elevated in plasma of preeclamptic women, and a 2-fold elevation of plasma TNF increases vascular resistance and arterial pressure in pregnant rats, suggesting a role of the cytokine in pregnancy-induced hypertension. However, whether the hemodynamic effects of TNF reflect direct effects of the cytokine on the mechanisms of vascular reactivity is unclear. The purpose of this study was to test the hypothesis that TNF directly impairs endothelium-dependent relaxation and enhances vascular reactivity in systemic vessels of pregnant rats. Active stress was measured in aortic strips isolated from virgin and late pregnant Sprague-Dawley rats and untreated or treated for 2 hr with increasing concentrations of TNF (10 to 1000 pg/ml). In endothelium intact vascular strips, phenylephrine (Phe) caused increases in active stress that were smaller in pregnant than virgin rats. TNF caused an enhancement of Phe contraction that was more prominent in pregnant than virgin rats. Acetylcholine (ACh) caused relaxation of Phe contraction and increases in vascular nitrite/nitrate production that were greater in pregnant compared to virgin rats. TNF caused significant inhibition of ACh-induced vascular relaxation and nitrite/nitrate production that were more prominent in pregnant than virgin rats. Incubation of endothelium-intact strips in the presence of L-NAME (100 µM), to inhibit nitric oxide (NO) synthase, or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 1 µM), to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in untreated but to a less extent in TNF-treated vessels particularly those of pregnant rats. Removal of the endothelium enhanced Phe-induced stress in untreated but not TNF-treated vessels particularly those of pregnant rats. In endothelium-denuded strips, relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was not significantly different between untreated and TNF-treated vessels of virgin or pregnant rats. Thus TNF enhances vascular reactivity and inhibits an endothelium-dependent NO-cGMP mediated vascular relaxation pathway in systemic vessels particularly those of pregnant rats. The greater TNF-induced inhibition of vascular relaxation and enhancement of vascular reactivity in systemic vessels of pregnant rats supports a direct role for TNF as one possible mediator of the increased vascular resistance associated with pregnancy-induced hypertension.