Descending vasa recta (DVR) are 15 µm vessels that perfuse the renal medulla. Ouabain has been shown to augment DVR endothelial cytoplasmic Ca²⁺ ([Ca²⁺]_CYT) signaling. In this study we examined the expression of the ouabain sensitive NaKATPase 2 subunit in the rat renal vasculature and tested effects of acute ouabain exposure and chronic ouabain treatment on DVR. Immunostaining with antibodies directed against the 2 subunit verified its expression in both DVR pericytes and endothelium. Acute application of ouabain (100 nM or 500 nM) augmented the DVR NO (nitric oxide) generation stimulated by acetylcholine (ACh, 10 µM). At a concentration of 1 mM, ouabain constricted microperfused DVR, whereas at 100 nM, it was without effect. Acute ouabain (100 nM) did not augment constriction by angiotensin II (0.5 or 10 nM), whereas L-NAME (L-nitroarginine methyl ester) induced contraction of DVR was slightly enhanced. Ouabain hypertensive (OH) rats were generated by chronic ouabain treatment (30 µg/Kg-day, 5 weeks). The acute endothelial [Ca²⁺]_CYT elevation by ouabain (100 nM) was absent in DVR endothelia of OH rats. The [Ca²⁺]_CYT response to 10 nM acetylcholine was also eliminated whereas the response to 10 µM acetylcholine was not. The endothelial [Ca²⁺]_CYT response to bradykinin (100 nM) was significantly attenuated. We conclude that endothelial responses may offset the ability of acute ouabain exposure to enhance DVR vasoconstriction. Chronic exposure to ouabain, in vivo, leads to hypertension and DVR endothelial dysfunction, manifest as reduced [Ca²⁺]_CYT responses to both ouabain and endothelium dependent vasodilators.