Prolonged ouabain administration (25 μg/kg/day, 5 wk) induces 'ouabain hypertension' (OH) in rats, but the molecular mechanisms by which ouabain elevates blood pressure are unknown. Here, we compare Ca²⁺ signaling in mesenteric artery smooth muscle cells (ASMCs) from normotensive (NT) and OH rats. Resting cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt measured with Fura-2) and phenylephrine-induced Ca²⁺ transients were augmented in freshly-dissociated OH ASMCs. Immunoblots revealed that expression of ouabain-sensitive 2 Na⁺ pumps, but not the predominant, ouabain-resistant 1 pumps, was increased (2.5-fold NT), as was Na/Ca exchanger-1, NCX1 (6-fold NT) in OH arteries. Ca²⁺ entry, activated by sarcoplasmic reticulum (SR) Ca²⁺ store depletion with cyclopiazonic acid (SR Ca²⁺-ATPase inhibitor) or caffeine, was augmented in OH ASMCs. This reflected 2.5-fold augmented expression, in OH ASMCs, of TRPC1, an essential component of store-operated channels (SOCs); two other components of some SOCs were not expressed (TRPC4) or not up-regulated (TRPC5). Ba²⁺ entry activated by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (a measure of receptor-operated channel, ROC, activity) was much greater in OH than NT ASMCs. This correlated with 6-fold up-regulation of TRPC6 protein, a ROC family member. Importantly, in primary cultured mesenteric ASMCs from normal rats, 72 h treatment with 100 nM ouabain significantly augmented NCX1 and TRPC6 protein expression and increased resting [Ca²⁺]_cyt and ROC activity. SOC activity was also increased. Silencer (si)RNA knockdown of NCX1 markedly down-regulated TRPC6 and eliminated the ouabain-induced augmentation; siRNA knockdown of TRPC6 did not affect NCX1 expression, but greatly attenuated its up-regulation by ouabain. Clearly, NCX1 and TRPC6 expression are interrelated. Thus, prolonged ouabain treatment up-regulates the 2 Na⁺ pump-NCX1-TRPC6 (ROC) Ca²⁺ signaling pathway in arterial myocytes in vitro as well as in vivo. This may explain the augmented myogenic responses and enhanced phenylephrine-induced vasoconstriction in OH arteries (83) as well as the high blood pressure in OH rats.