Offspring of mothers fed a high fat diet during pregnancy show an increased incidence of diabetes, hypertension, obesity and vascular dysfunction with reduced endothelium-dependent nitric oxide (NO) and hyperpolarisation (EDH)-dependent vasodilation .The current study further investigates the impact of a maternal high fat diet on the EDH-mediated vasodilator signalling pathways, Female C57/BL6 mice were fed either a high fat (HF, n=21) or control diet (C, n=17) 6 weeks prior to conception and throughout gestation and lactation. On weaning, offspring were fed the same or opposite diet to their dams to give four offspring groups: C/C, C/HF, HF/C and HF/HF. Offspring bodyweight, plasma glucose and blood pressure were measured at 15 weeks of age, prior to culling by cardiac exsanguination. Sections of mesenteric arterioles (0.2 mm in length) were imaged using confocal microscopy and the endothelial expression and localization of intermediate conductance calcium activated potassium channel (IKCa) and connexin 37 quantified. Results are expressed as mean ± SEM (C/C n= 5, C/HF n=4, HF/C n=5, HF/HF n=3) and analysed by Student’s t-test and one-way ANOVA with Dunnett’s multiple comparisons. HF dams were heavier than C dams (C 27.7g ± 0.7 vs HF 37.83g ± 1.46 p<0.0001) with higher plasma glucose (C 7.6mmol/L ± 1.3 vs HF 10.9mmol/L ± 1.3, p<0.05) and abdominal adiposity (C 0.32g ± 0.06 vs HF 1.15g ± 0.23 p<0.05). While C/HF and HF/HF offspring were heavier than C/C and HF/C offspring (C/C 30.3g ± 2.7, C/HF 38.3g ± 7.6, HF/C 32.2g ± 5.3, HF/HF 41.9g ± 6.5, p<0.0001) there was no significant difference in systolic blood pressure between the groups. Maternal HF-diet influenced endothelial cell IKCa expression (%; C/C 22.5 ± 4.6, C/HF 20.0 ± 1.2, HF/C 25.9 ± 2.7, HF/HF 45.1 ± 11.3 p<0.05). Colocalization of IKCa to the endothelial cell nucleus was also increased in the HF/C group (Mander’s overlap coefficient; C/C 0.58 ± 0.05, C/HF 0.50 ± 0.02, HF/C 0.72 ± 0.01, HF/HF 0.62 ± 0.05, p<0.05.) Expression of connexin 37 and the colocalization of connexin 37 and IKCa were not significantly influenced by maternal or post-weaning diet. These data are consistent with an altered contribution of EDH-signalling in the vasodilatation of small vessels in offspring from HF-fed dams and are indicative that an adverse developmental environment may impact on the normal growth and maturational-related changes in endothelial function on which pathological changes may be superimposed to produce an ‘at risk’ phenotype in the adult offspring.