Some "in vivo" and "ex vivo" studies demonstrated a resistance to the vasodilating effects of nitric oxide (NO) in insulin resistant states, and, in particular, in obese Zucker rats (OZR).To evaluate the biochemical basis of this phenomenon, we aimed to identify defects of the NO/cGMP/PKG pathway in cultured Vascular Smooth Muscle Cells (VSMC) from OZR and lean Zucker rats (LZR),by measuring: i)NO donor ability to increase cGMP in the absence and in the presence of inhibitors of soluble guanylate cyclase (sGC) and phosphodiesterases (PDEs); ii)NO and cGMP ability to induce -via PKG- VASP phosphorylation at Serine 239 and PDE5 activity; iii)protein expression of sGC, PKG, total VASP and PDE5; iv)superoxide anion concentrations and ability of antioxidants (SOD+Catalase and Amifostine) to influence the NO/cGMP/PKG pathway activation; v)hydrogen peroxide influence on PDE5 activity and VASP phosphorylation. VSMC from OZR vs LZR showed: i)baseline cGMP concentrations higher, at least in part owing to reduced catabolism by PDEs; ii)impairment of NO donor ability to increase cGMP even in the presence of PDE inhibitors, suggesting a defect in the NO-induced sGC activation; iii)reduction of NO and cGMP ability to activate PKG, indicated by the impaired ability to phosphorylate VASP at Serine 239 and to increase PDE5 activity via PKG; iv)similar baseline protein expression of sGC, PKG, total VASP and PDE5; v)higher levels of superoxide anion. Antioxidants partially prevented the defects of NO/cGMP/PKG pathway observed in VSMC from OZR, that were reproduced by hydrogen peroxide in VSMC from LZR, suggesting the pivotal role of oxidative stress.