Hyperhomocysteinemia (HHcy) impairs endothelium-dependent vasodilation by increasing reactive oxygen species thereby reducing nitric oxide (NO·) bioavailability. It is unclear whether reduced expression or function of the enzyme that produces NO·, eNOS, also contributes. It is also unclear whether resistance vessels undergo alteration of non-NO· mechanisms in this condition. We tested these hypotheses in male C57BL/6 mice with chronic HHcy induced by 6-wk high methionine/low-B vitamin feeding (Hcy: 89.2 ± 49.0 µM) compared to age-matched controls (Hcy: 6.6 ± 1.9 µM), using first order mesenteric arteries. Dilation to acetylcholine (ACh, 10^-9 - 10^-4 M) was measured in isolated, cannulated, pressurized (75 mmHg) arteries with and without L-NAME (10^-4 M) and/or indomethacin (10^-5 M) to test endothelium-dependent dilation and non-NO dependent dilation, respectively. The time-course of dilation to ACh (10^-4 M) was examined to compare the initial transient dilation due to non-NO, non-prostacyclin mechanism and the sustained dilation due to NO·. These experiments indicated that endothelium-dependent dilation was attenuated (P<0.05) in HHcy arteries due to down-regulation of only NO·-dependent dilation. Western blotting indicated significantly less (P<0.05) basal eNOS and phospho-S1179-eNOS/eNOS in mesenteric arteries from HHcy mice, but no difference in phospho-T495-eNOS/eNOS. S1179 eNOS phosphorylation was also significantly less in these arteries when stimulated with ACh ex vivo or in situ. Real-time PCR indicated no difference in eNOS mRNA levels. In conclusion, chronic diet-induced HHcy in mice impairs eNOS protein expression and phosphorylation at S1179, coincident with impaired NO·-dependent dilation, which implicates dysfunction in eNOS post-transcriptional regulation in the impaired endothelium-dependent vasodilation.