Female Wistar rats were mated and fed diets that altered in protein, folate or methionine content throughout pregnancy. In both the male and female PR offspring no difference to the control offspring in systolic blood pressure was found by tail-cuff plethysmography. However, endothelium-dependent relaxation of the small mesenteric arteries as assessed by small wire myography was impaired (p<0.05), with a specific reduction in the NO component of this relaxation response. There was also remodelling of these arteries with a reduction in smooth muscle content. In the PR male offspring, the mRNA expression of endothelial nitric oxide synthase (eNOS) as measured by real-time PCR was increased in the mesenteric arteries (p<0.05) but the level of soluble guanylate cyclase was unchanged. mRNA expression of the antioxidants manganese superoxide dismutase and glutamate cysteine ligase was unchanged with maternal diet but the expression of heme oxygenase-1 (HO-1) was reduced (p<0.05) in the PR males' livers. However, the level of oxidative stress as measured by protein carbonyl concentration in the livers was not found to be altered following maternal PR. Neither maternal folate supplementation nor the partial removal of methionine from the maternal diet prevented the endothelial dysfunction in the PR male offspring. Thus, PR in utero alters the structure and function of the resistance arteries of both the male and female offspring, which results from both an attenuated NO pathway and a reduced antioxidant defence compensated for in the males by up-regulation of eNOS mRNA expression. This study provides a mechanism for the induction of vascular dysfunction by the pre-natal environment, which may be relevant to the developmental origins of CVD.