Rationale: Maternal obesity pre-programmes offspring to develop obesity and associated cardiovascular disease. Perivascular adipose tissue (PVAT) exerts an anti-contractile effect on the vasculature, which is reduced in hypertension and obesity. Objective: To determine whether maternal obesity pre-programmes offspring to develop PVAT dysfunction in later life. Methods: Female Sprague-Dawley rats were fed a diet containing 10% (control) or 45% fat (HFD) for 12 weeks prior to mating and during pregnancy and lactation. Male offspring were killed at 12 or 24 weeks of age and tension in PVAT-intact or -denuded mesenteric artery segments measured isometrically. Concentration-response curves were constructed to U46619 and norepinephrine. Results: Only 24 week old HFD offspring were hypertensive (p<0.0001), although the anticontractile effect of PVAT was lost in vessels from HFD offspring of each age. Inhibition of nitric oxide (NO) synthase with 100 μM L-NMMA attenuated the anti-contractile effect of PVAT and increased contractility of PVAT-denuded arteries (p<0.05, p<0.0001). The increase in contraction was smaller in PVAT-intact than PVAT-denuded vessels from 12 week old HFD offspring, suggesting decreased PVAT-derived NO and release of a contractile factor (p<0.07). An additional, NOindependent effect of PVAT was evident only in norepinephrine-contracted vessels. Activation of AMP-activated kinase (with 10μM A769662) was anti-contractile in PVAT-denuded (p<0.0001) and –intact (p<0.01) vessels and was due solely to NO in controls; the AMPK effect was similar in HFD offspring vessels (p<0.001 and p<0.01, respectively) but was partially NO-independent. Conclusions: The diminished anti-contractile effects of PVAT in offspring of HFD dams are primarily due to release of a PVAT-derived contractile factor and reduced NO bioavailability.