OBJECTIVE: We sought to describe the effects of ouabain on the human uteroplacental vasculature. STUDY DESIGN: Stem villous vessels and intramyometrial arteries isolated from placental and myometrial biopsy specimens at term were mounted in organ baths. Moreover, isolated human placental cotyledons were perfused. RESULTS: Contractions induced by the thromboxane A₂ analog U46619 were unaffected by pretreatment with ouabain 10^-10 to 10^-6 mol/L. In fetal vessels nitric oxide (10^-8 to 3 x 10^{- 5} mol/L) induced relaxation of vascular tonus induced by U46619 and potassium. This relaxation was inhibited by pretreatment with ouabain 10^-7 to 10^-6 mol/L. These associations were unaffected by removal of the endothelium. In maternal arteries ouabain (10^-6 mol/L) failed to significantly affect nitric oxide-induced relaxation. Ouabain (10^-9 mol/L) significantly affected pressure-flow relationships in perfused cotyledons. CONCLUSIONS: Ouabain impairs nitric oxide-induced relaxation of human stem villous arteries and veins, which may explain the changes induced by therapeutically relevant concentrations of the drug on pressure-flow relationships in the perfused cotyledon. Thus treatment with cardiac glycosides in pregnancy may impair uteroplacental blood flow.