Ca2+ is essential for endothelial production of vasorelaxing factors. We determined whether the impaired endothelium-dependent relaxation in aldosterone-salt hypertensive rats (AHR) is associated with a decreased free Ca2+ ([Ca2+]i) response in endothelial cells. In isolated aorta, the EC50 for the acetylcholine-induced endothelium-dependent relaxations did not differ between AHR and the age-matched control-salt rats (CSR). However, maximal relaxation was significantly reduced by 47% in AHR (P < .05). In contrast, the endothelium-independent relaxation to sodium nitroprusside was not impaired in aorta from AHR. The [Ca2+]i was measured with fura-2 microfluorometry in endothelial cells freshly dispersed from aorta. Although the basal [Ca2+]i was not different between CSR and AHR, the peak [Ca2+]i response to acetylcholine was significantly reduced in cells from AHR compared with CSR (P < .05). These results suggest that depressed endothelial [Ca2+]i responses to acetylcholine may be involved in the impaired endothelium-dependent relaxation in aorta from AHR.