There is increasing evidence that poor early growth confers an increased risk of developing type 2 diabetes, hypertension, and other features of the metabolic syndrome in later life. It is hypothesized that this may result from a mismatch during early life exerting permanent effects on the structure and function of key metabolic organ systems, including the vasculature. The aim of this study was to explore the long-term impact of a dietary mismatch on endothelial function and specifically to investigate whether a decreased bioavailability of nitric oxide (NO) is a primary feature of endothelial dysfunction in this model. Mice (C57BL/J) were exposed to either a high fat (HF, 45% kcal fat) or standard animal chow (C, 21% kcal fat) diet throughout pregnancy and lactation. The male offspring were then continued on the same diet as their dams or subjected to a nutritional mismatch and crossed over onto the alternative diet, for 10 to 12 weeks post weaning, to give four dietary study groups (C/C, C/HF, HF/C and HF/HF) comprising 10 animals per group. Blood pressure (tail cuff plethysmography, SBP average 6 measures) and body weight were recorded at weekly intervals. Femoral artery vasorelaxation to ACh (1nM-10μM) was assessed using wire myography and basal NO production using 4,5-diaminofluoresceine diacetate (DAF-2 DA), an NO-sensitive fluorescent dye. Vascular segments were incubated for 45 min at 37°C with 5 µM DAF-2 DA in HEPES buffer (pH 7.4) and digital images collected using confocal microscopy (excitation at 490 nm; emission 535 nm). The images were analyzed using image software by measuring the mean OD of the fluorescence observed in the endothelium. Offspring fed the HF diet post weaning (HF/HF and C/HF groups) gained more weight than the C/C and HF/C animals. Blood pressure was also significantly higher in the HF/HF and C/HF. Arteries from HF/HF and C/HF animals showed an impaired endothelium-dependent relaxation to ACh compared with C/C and HF/C animals (P<0.05). Basal NO production was greater in CC arteries compared with HF/HF, with staining most evident in the inter-junctional regions of the endothelium and in the underlying intima. Our preliminary data demonstrate the impact of an in utero and postnatal HF diet on vascular function and suggest that the endothelial dysfunction observed is a result of impairment of NO production and/or bioavailability. It further suggests that both predisposition to disease, acquired in early life, and later lifestyle may contribute to the development of cardiovascular disease which are sustained into adulthood.