.-Previously, using an animal model of syndrome X, the obese Zucker rat (OZR), we documented impaired endothelium-dependent vasodilation. The aim of this study was to determine whether reduced expression or altered posttranslational regulation of endothelial nitric oxide synthase (eNOS) underlies the vascular dysfunction in OZR rats. There was no significant difference in the relative abundance of eNOS in hearts, aortas, or skeletal muscle between lean Zucker rats (LZR) and OZR regardless of age. There was no difference in eNOS mRNA levels, as determined by real-time PCR, between LZR and OZR. The inability of insulin resistance to modulate eNOS expression was also documented in two additional in vivo models, the ob/ob mouse and the fructose-fed rat, and in vitro via adenoviral expression of protein tyrosine phosphatase 1B in endothelial cells. We next investigated whether changes in the acute posttranslational regulation of eNOS occurs with insulin resistance. Phosphorylation of eNOS at S632 (human S633) and T494 was not different between LZR and OZR; however, phosphorylation of S1176 was significantly enhanced in OZR. Phosphorylation of S1176 was not different in the ob/ob mouse or in fructose-fed rats. The association of heat shock protein 90 with eNOS, a key regulatory step controlling nitric oxide and aberrant O 2 Ϫ production, was not different between OZR and LZR. Taken together, these results suggest that changes in eNOS expression or posttranslation regulation do not underlie the vascular dysfunction seen with insulin resistance and that other mechanisms, such as altered localization, reduced availability of cofactors, substrates, and the elevated production of O 2 Ϫ , may be responsible. syndrome X; Zucker; obesity INSULIN RESISTANCE, a prediabetic condition associated with obesity, is increasing at an alarming rate in Western cultures (26,30). Diabetes increases the incidence of many vasculopathies, including atherosclerosis, microvascular disease, and poor wound healing (44, 47). Of even greater concern is the mounting evidence that insulin-resistant states, independent of frank diabetes, also promote vascular dysfunction (43, 52). Thus the effect of insulin resistance on vascular signaling mechanisms is an area of increasing scrutiny. Insulin-resistant states and diabetes are associated with reduced endothelium-dependent relaxation, and this dysfunction of the endothelium is highly correlated with future cardiovascular events (1,14,34,49). In the obese Zucker rat (OZR), we previously showed that endothelium-dependent relaxation is impaired (14); however, the mechanisms underlying reduced synthesis or action of nitric oxide (NO) remain to be established.Endothelium-dependent relaxation is mediated by the synthesis and subsequent diffusion of NO from the endothelium to the underlying smooth muscle (21, 35). Of the three NO synthases (NOS), the endothelial isoform (eNOS) is uniquely positioned within the vascular endothelium and alone is responsible for endothelium-dependent NO-mediated relaxation (20)....