Epidemiological studies have shown increased incidence of hypertension and coronary artery disease in growth-restricted fetuses during their adult life. A novel animal model was used to test the hypothesis regarding the role of abnormal uterine environment in fetal programming of adult vascular dysfunction. Mice lacking a functional endothelial nitric oxide synthase (NOS3^-/-KO) and wild-type mice (NOS3^+/+WT) were cross bred to produce homozygous NOS3^-/-KO, maternally-derived heterozygous (NOS3^+/-mat, mother with NOS3 deficiency), paternally-derived heterozygous (NOS3^+/-pat, normal mother) and NOS3^+/+WT litters. Number of fetuses per litter was smaller in NOS3^-/-KO and NOS3^+/-mat compared with NOS3^+/-pat and NOS3^+/+WT. Adult female mice from these litters (7-8 weeks old) were sacrificed and rings preparations of carotid and mesenteric arteries were mounted in a wire myograph to evaluate the passive and reactive vascular characteristics. Slope of the length-tension plot (a measure of vascular compliance) was increased and optimal diameter (as calculated by Laplace equation) was decreased in NOS3^-/-KO and NOS3^+/-mat compared with NOS3^+/-pat and NOS3^+/+WT. Acetylcholine caused vasorelaxation in NOS3^+/-pat and NOS3^+/+WT and contraction in NOS3^-/-KO and NOS3^+/-mat. Responses to phenylephrine and Ca²⁺ were increased in NOS3^-/-KO and NOS3^+/-mat compared to NOS3^+/-pat and NOS3^+/+WT. Relaxation to isoproterenol was decreased in NOS3^-/-KO and NOS3^+/-mat versus NOS3^+/-pat and NOS3^+/+WT. Abnormalities in the passive and reactive in-vitro vascular properties seen in NOS^+/-mat mice that developed in a NOS3 deficient maternal/uterine environment compared to the genetically identical NOS3^+/-pat mice developing in a normal environment provides the first direct evidence in support of a role for uterine environment in determining vascular function in later life.