Apolipoprotein-E knockout (ApoE-/-) mice develop hypercholesterolemia and are a good model of atherosclerosis. Changes in Ca2+ are vital to the function of endothelial cells within vessels, but understanding of how atherosclerosis changes these signals, especially early in the disease, is limited. We have examined the effect of hypercholesterolemia on carbachol-mediated Ca2+ signals in aortic endothelial cells from wild-type (WT) and ApoE-/- mice of different ages; 10 weeks, before overt plaques are present and 24 weeks, where plaques are well established. The extent of plaque development was confirmed using en face staining with Sudan IV. Using confocal microscopy (Fluo-4-AM loading) allowed us to distinguish clearly the Ca2+ signals emanating from the endothelial cells alone, with the endothelium:smooth muscle interface intact. In addition, we compared the CCh response in thoracic aorta and in the more plaque-prone aortic arch. Three parameters of the Ca2+ responses were measured; the size of the initial Ca2+ peak, the size of the secondary plateau phase and area under the curve (AUC). Significance was assessed using the Students t-test. All aortae from older ApoE-/- mice exhibited clear atherosclerotic plaques (n=9), whereas aortae from 10 week old ApoE-/- mice showed either no plaque or barely visible tiny areas indicating where plaques would later form (n=10). No WT mice exhibited atherosclerotic plaques in the aorta (n=5-10). In all tissues, CCh produced a concentration-dependent increase in all 3 parameters of the intracellular Ca2+ response. The aortic arch is more prone to development of atherosclerotic plaques than the thoracic aorta and it is this region in ApoE-/- mice that demonstrated the greatest alteration in CCh-mediated Ca2+ signalling. Thus, in aortic arch from 24 week old ApoE-/- mice, a significantly increased Ca2+ response was seen to all CCh concentrations (0.3-10μM) compared to WT, measured as peak, plateau or AUC responses (n=4-6 mice). In aortic arch from 10 week olds, the dose-response curves for peak Ca2+ were identical in WT and ApoE-/-, however for the plateau and AUC responses, again a larger Ca2+ response was observed in the ApoE-/- mice, though this time the difference was seen at lower concentrations (1 & 3μM, n=4-5 mice). In thoracic aorta from young and older mice, a significant difference was only seen between WT and ApoE-/- responses at the highest concentrations of CCh, with the ApoE-/- mice showing a greater peak, plateau and AUC response to 10μM CCh. Therefore there was no age-dependent alteration in response in thoracic aorta. It appears that hypercholesterolemia alone is enough to modify the CCh response in both thoracic and arch sections of ApoE-/- aortae, though the presence of atherosclerotic plaques causes a much more significant change in Ca2+ signalling.