The long-term benefits of nitroglycerin therapy are limited by the tolerance development. Understanding of the precise nature of mechanisms underlying nitroglycerin-induced endothelial cell dysfunction may provide new strategies to prevent the tolerance development. In this line, we tested the interventions to prevent endothelial dysfunction in the setting of nitrate tolerance. When bovine aortic endothelial cells (BAECs) were continuously treated with nitric oxide (NO) donors, including nitroglycerin, over 2-3 days, basal production of nitrite and nitrate (NOx) was diminished. The diminished basal NOx levels were mitigated by intermittent treatment, allowing for an 8-h daily nitrate-free interval during the 2-3-day treatment period. The addition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin restored the basal levels of NOx that were decreased by continuous nitroglycerin treatment of BAECs. Apocynin caused significant improvement of increased mRNA and protein levels of endothelial NO synthase (eNOS) in BAECs given nitroglycerin continuously over the treatment period. Apocynin also reduced endothelial production of reactive oxygen species (ROS) following continuous nitroglycerin treatment. These results showed an essential similarity to the effects of a nitrate-free interval. Application of the NOS inhibitor N-nitro-L-arginine methyl ester caused a recovery effect on basal NOx and eNOS expression but was without effect on ROS levels in continuously NO donor-treated BAECs. In conclusion, the present study characterized abnormal features and functions of endothelial cells following continuous NO donor application. We suggest that inhibition of NADPH oxidase, by preventing NO donor-induced endothelial dysfunction, may represent a potential therapeutic strategy that confers protection from the nitrate tolerance development.