The purpose was to investigate the role of calcium (Ca2+) for agonist-induced NO release in the rat SMA and HUVEC. Acetylcholine (ACh) increased the NO concentration and relaxed the arteries. In Ca2+-free solution ACh did not increase NO concentration. An inhibitor of the SR calcium-ATPase cyclopiazonic acid (CPA) induced a sustained increase in NO concentration in the presence of Ca2+, but a transient increase in the absence of Ca2+. In the presence of a combination of apamin (Apa) and charybdotoxin (ChTx), ACh-induced increases in NO concentration were blunted, and CPA only evoked transient increases. In everted arterial preparations, ACh and CPA increased FURA-2 ratio and relaxed arteries with endothelium, but these response were absent in preparations without endothelium. Nifedipine did not change increases in FURA-2 ratio, but the presence of the blocker of store-operated Ca2+ channels, SKF-96365, blocked ACh and CPA-induced increases in FURA-2 ratio. Apa and ChTx did not change ACh-evoked increases in FURA-2 ratio. In HUVEC histamine increased the NO concentration and [Ca2+], which were reduced in the presence of Apa and ChTx. The findings suggest that K+Ca channels are coupled to increased Ca2+ influx and formation of NO in isolated endothelial cells, while K+Ca channels regulate NO release without changing endothelial Ca2+ in intact arteries.