The endothelium is the innermost lining of blood vessels and plays a significant role in controlling virtually every cardiovascular function. Constant signalling among endothelial cells and between endothelial cells and blood cells and smooth muscle cells is critical for the control of blood fluidity, vascular tone and angiogenesis. Intracellular Ca2+ signals act as a messenger system that decodes extracellular signals arriving at the endothelium and encodes intracellular signals to determine cardiovascular activity. Hydrogen peroxide (H2O2), a key reactive oxygen species (ROS) generated by the respiratory chain in mitochondria, is also an important regulator of endothelial function. In endothelial cells, H2O2 may also be produced by NADPH oxidase, xanthine oxidase and nitric oxide synthases. Although mitochondria‐derived ROS are usually considered to induce oxidative stress and damage cells, the physiological roles of ROS in mechano‐stress signal transduction, vascular relaxation, permeability of endothelial cells is now acknowledged. In this study, the interactions of low, physiological, concentrations (1 μM) H2O2 and Ca2+ signals were examined in the endothelium of intact rat mesenteric arteries. The intracellular Ca2+ was visualized and measured in ~200 endothelial cells using the Ca2+ indicator Cal‐520 through high‐resolution, high‐speed imaging. Endothelial cells responded to ACh (100 nM) with Ca2+ signals that initiated in clusters of endothelial cells and propagated within and between cells. ACh‐evoked Ca2+ signals were largely IP3‐evoked signalling events since the signals persisted after removal of external Ca2+ and were blocked by the SERCA inhibitor cyclopiazonic acid (10 μM) and channel blocker 2‐APB (100 μM). H2O2 (100 nM – 1 mM) inhibited ACh‐evoked Ca2+ signals in a concentration‐dependent manner. Low concentrations (e.g. 1 μM) of H2O2 are likely to be most physiologically relevant in signalling and were investigated further. H2O2 suppression of ACh‐evoked Ca2+ signals appeared as a decrease in the amplitude of the Ca2+ signal in each activated cell and a reduced number of cells activated. H2O2 inhibition of Ca2+ signals were reversed by catalase (1000 U/ml). H2O2 may decrease ACh‐evoked Ca2+ signals by reducing the affinity of the ACh receptor, decreasing the production of IP3 or limiting the activity of the IP3 receptor. To distinguish between these possibilities, the effects of H2O2 on direct activation of the IP3 receptor was examined using photoactivatable caged‐inositol 1,4,5‐trisphosphate (cIP3). Ca2+ release from IP3 receptor, by photolysis of cIP3, was inhibited by H2O2, an effect reversed by catalyse (1000 U/ml). Together, these results suggest low concentrations of H2O2 inhibit intracellular Ca2+ signalling by desensitizing IP3 receptors. These findings point an important interaction in low concentrations of ROS in the control of physiological Ca2+ signalling.Support or Funding InformationSupported by the Wellcome Trust and British Heart FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.