1 This study was undertaken to determine whether long-term in vivo administration of nitroglycerine (NTG) downregulates the endothelium-dependent relaxation induced by acetylcholine (ACh) in the rabbit intrapulmonary vein and, if so, whether the type 1 angiotensin II receptor (AT(1)R) blocker valsartan normalizes this downregulated relaxation. 2 In strips treated with the cyclooxygenase inhibitor diclofenac, ACh induced a relaxation only when the endothelium was intact. A small part of this ACh-induced relaxation was inhibited by coapplication of two Ca(2+)-activated K(+)-channel blockers (charybdotoxin (CTX)+apamin) and the greater part of the response was inhibited by the nitric-oxide-synthase inhibitor N(omega)-nitro-L-arginine (L-NNA). 3 The endothelium-dependent relaxation induced by ACh, but not the endothelium-independent relaxation induced by the nitric oxide donor NOC-7, was significantly reduced in NTG-treated rabbits (versus those in NTG-nontreated control rabbits). The attenuated relaxation was normalized by coapplication of valsartan with the NTG. 4 In the vascular wall, both the amount of localized angiotensin II and the production of superoxide anion were increased by in vivo NTG treatment. These variables were normalized by coapplication of valsartan with the NTG. 5 It is suggested that long-term in vivo administration of NTG downregulates the ACh-induced endothelium-dependent relaxation, mainly through an inhibition of endothelial nitric oxide production in the rabbit intrapulmonary vein. A possible role for AT(1)R is proposed in the mechanism underlying this effect.