Nitric oxide (NO)-induced vasorelaxation and the regulation of endothelial superoxide anion levels is partly mediated by vascular large conductance Ca 2 + -activated K + (BK C a ) channels. Nitroglycerin acts through the release of NO and its effect is modulated by changes in endothelial superoxide levels. This study examines the effect of BK C a channel blockade on nitroglycerin-induced vasorelaxation in human arterial and venous vascular segments and whether responses to BK C a channel blockade are influenced by the development of venous nitroglycerin tolerance. Dose-relaxation curves to nitroglycerin (10 - 1 0 -10 - 4 M) were obtained in segments of the saphenous vein and the left mammary artery. Studies were performed with and without pre-incubation with the BK C a channel blocker iberiotoxin (10 - 7 M) and venous tolerance to nitroglycerin were induced by a 24-h i.v. infusion (0.5 μg/kg/min). Iberiotoxin reduced the vasorelaxant effect of nitroglycerin (E m a x ) by 60% in endothelium-intact arteries and 13% in endothelium-denuded arteries (P<0.05). Development of nitroglycerin tolerance did not affect the response to iberiotoxin in the venous vascular segments (P>0.05) and (compared to arterial segments) veins were less sensitive to BK C a channel blockade (30% reduction in E m a x ) or endothelial removal. The results suggest that primarily arterial effects of nitroglycerin are significantly inhibited by changes in the activity of the endothelial BK C a channels. Although endothelial BK C a are likely regulators of mechanisms underlying arterial tolerance development to nitroglycerin, they do not appear to play a role in human venous nitroglycerin tolerance development.