NO, constitutively produced by endothelial NO synthase (eNOS), plays a key regulatory role in vascular wall homeostasis. We generated transgenic (Tg) mice overexpressing eNOS in the endothelium and reported the presence of reduced NO-elicited relaxation. The purpose of this study was to clarify mechanisms of the reduced response to NO-mediated vasodilators in eNOS-Tg mice. Thoracic aortas of Tg and control mice were surgically isolated for vasomotor studies. Relaxations to acetylcholine and sodium nitroprusside were significantly reduced in Tg vessels compared with control vessels. Relaxations to atrial natriuretic peptide and 8-bromo-cGMP were also significantly reduced in Tg vessels. Reduced relaxations to these agents were restored by chronic N(G)-nitro-L-arginine methyl ester treatment. Basal cGMP levels of aortas were higher in Tg mice than in control mice, whereas soluble guanylate cyclase (sGC) activity in Tg vessels was approximately 50% of the activity in control vessels. Moreover, cGMP-dependent protein kinase (PKG) protein levels and PKG enzyme activity were decreased in Tg vessels. These observations indicate that chronic overexpression of eNOS in the endothelium resulted in resistance to the NO/cGMP-mediated vasodilators and that at least 2 distinct mechanisms might be involved: one is reduced sGC activity, and the other is a decrease in PKG protein levels. We reported for the first time that increased NO release from the endothelium reduces sGC and PKG activity in mice. These data may provide a new insight into the mechanisms of nitrate tolerance and cross tolerance to nitrovasodilators.