Chronic administration of Nω-nitro-L-arginine methyl ester (L-NAME) in rats is a chemical method to study the induction and progression of nitric oxide (NO) deficiency-induced endothelial dysfunction. Male Wistar rats received L-NAME (50 mg/kg/day in drinking water) or no drug for 6 weeks. Mean arterial pressure (MAP) was measured on Day 43 by carotid artery cannulation. Plasma interleukin 1β (IL-1β) level was measured by enzyme-linked immunosorbent assay. Aorta and carotid artery were isolated for determination of basal nitrite, cGMP production, soluble guanylylcyclase (sGC) activity, phosphodiesterase-5 (PDE5) activity, and dimethylarginine dimethylaminohydrolase (DDAH) activity. mRNA expression studies were done by real time-polymerase chain reaction. L-NAME induced an increase in MAP and plasma IL-1β. The treatment had varied effect on endothelial nitric oxide synthase (eNOS), sGC, and PDE5 but showed an increase in inducible NOS (iNOS) mRNA expression and plasma asymmetric dimethyl arginine levels. Basal nitrite, cGMP levels, sGC activity, and DDAH activity were significantly decreased in the tissues. Brief incubation of tissues in vitro with 1400 W, a specific iNOS blocker, partially reversed sGC activity, and cGMP levels. The results of this study showed that L-NAME-mediated inhibition of eNOS is only partially responsible for the vascular pathology observed in this model. Secondary effects that include an increase in iNOS and a decrease in DDAH activity are likely to be the causative factors for the progression of vascular dysfunction.