Vascular endothelial cells express the ligand-activated transcription factor, peroxisome proliferator-activated receptor-gamma (PPARgamma), which participates in the regulation of metabolism, cell proliferation, and inflammation. PPARgamma ligands attenuate, whereas the loss of function mutations in PPARgamma stimulate, endothelial dysfunction, suggesting that PPARgamma may regulate vascular endothelial nitric oxide production. To explore the role of endothelial PPARgamma in the regulation of vascular nitric oxide production in vivo, mice expressing Cre recombinase driven by an endothelial-specific promoter were crossed with mice carrying a floxed PPARgamma gene to produce endothelial PPARgamma null mice (ePPARgamma(-/-)). When compared with littermate controls, ePPARgamma(-/-) animals were hypertensive at baseline and demonstrated comparable increases in systolic blood pressure in response to angiotensin II infusion. When compared with those of control animals, aortic ring relaxation responses to acetylcholine were impaired, whereas relaxation responses to sodium nitroprusside were unaffected in ePPARgamma(-/-) mice. Similarly, intact aortic segments from ePPARgamma(-/-) mice released less nitric oxide than those from controls, whereas endothelial nitric oxide synthase expression was similar in control and ePPARgamma(-/-) aortas. Reduced nitric oxide production in ePPARgamma(-/-) aortas was associated with an increase in the parameters of oxidative stress in the blood and the activation of nuclear factor-kappaB in aortic homogenates. These findings demonstrate that endothelial PPARgamma regulates vascular nitric oxide production and that the disruption of endothelial PPARgamma contributes to endothelial dysfunction in vivo.