Technical Abstract: The attenuation of endothelium-dependent nitric oxide (NO)-mediated vasodilation is a consistent finding in both conduit and resistance vessels during dietary Cu deficiency. While the effect is well established, evidence for the mechanism is still circumstantial. This study was designed to determine the relative amount of NO produced in and released from the vascular endothelium. Using the fluorescent NO indicator, DAF-FM, we now demonstrate the effect of a Cu-deficient diet on the production of NO from the endothelium of resistance arterioles. In one group of experiments, control and Cu-chelated lung microvascular endothelial cells (ECs) were used to assay NO production and fluorescence was observed by confocal microscopy. Weanling Sprague-Dawley rats were fed purified diets which were either Cu-adequate (6.3 'g Cu/g diet) or Cu-deficient (0.3'g Cu/g diet) for 4 weeks. In the second series of experiments, first-order arterioles were microsurgically isolated from the rat cremaster muscle, cannulated and pressurized with MOPS-PSS. DAF-FM (5 'M) was added in the lumen of the vessel to measure NO release. Baseline DAF-FM fluorescence was significantly less in Cu-chelated ECs compared to controls. In response to 10-6 M Ach, fluorescent intensity was significantly less in chelated ECs and in the lumen of Cu-deficient arterioles. The results suggest that production and release of NO by the vascular endothelium is inhibited by a restriction of copper. This inhibition may account for the attenuated vasodilation previously reported in Cu-deficient rats.