The molecular chaperone, heat shock protein 90 (Hsp90), acts as an intermediate in the signaling cascades leading to activation of endothelial nitric oxide synthase (eNOS). In this study, we examine the participation of this pathway in nitric oxide (NO)-dependent vasodilation in the rat mesentery in vitro. In normal animals, immunoprecipitation of eNOS from intact mesentery coimmunoprecipitates Hsp90 and, additionally, both eNOS and Hsp90 colocalize to the endothelial lining of mesenteric vessels. In the perfused mesenteric vasculature of normal animals, geldanamycin (GA), a specific inhibitor of Hsp90 signaling, attenuates ACh-dependent vasodilation but does not affect vasodilation in response to sodium nitroprusside. Next, studies were performed in animals with experimental portal hypertension induced by portal vein ligation (PVL). In PVL animals, NOS catalytic activity is markedly enhanced in mesenteric tissue and the perfused mesentery is hyporesponsive to the vasoconstrictor methoxamine (MTX). GA significantly potentiates MTX-induced vasoconstriction after PVL, thereby partially reversing the hyporeactivity to this agent exhibited in the mesenteric vasculature after PVL. These studies suggest that Hsp90 can act as a signaling mediator of NO-dependent responses in the mesenteric circulation and indicate that the excessive NO production observed in portal hypertension is mediated in part through Hsp90 signaling.