Atrial fibrillation (AF) is the most common heart arrhythmia and a major risk factor for thromboembolic stroke and hearth failure. Atrial pressure and/or volume overload are the common feature of the diseases that cause AF suggesting that mechanical stress has a major role in the pathogenesis of AF. The atrial endocardial endothelium (AEE) is the interface between the myocardium and the circulating blood but its role in the mechanotransduction in the atrium is unknown. We investigated the AEE responses to increased mechanical stretch by subjecting novel primary AEE cells to chronic mechanical stretch and evaluating a number of endpoints. The mechanical stretch conditions included cyclic stretch of 30cycles/min at 10% displacement. AEE cells were subjected to cyclic stretch for 0, ½, 1, 2, 4, and 24h. Changes in the cell shape with elongation, hypertrophy and alignment of the cells and their stress fibres in a direction perpendicular to the lines of stretch were first observed after 1 h of applied stretch. Immunostaining and WB analysis showed that after 4 h of stretch the cells exhibited significant downregulation of the EC specific cell-adhesion proteins VE-cadherin and PECAM-1, which is suggestive of changed EC permeability. TRPC6 and BKCa are stretch sensitive ion channels and important regulators of Ca2+-signalling in the EC cells. Altered distribution of the two channels was detected in the stretched AEE cells. Intracellular Ca2+ measurements in AEE cells at baseline and under stretch conditions showed decreased Ca2+ influx in stretched cells. These findings suggest that TRPC6 and BKCa may be important mediators of mechanical stretch responses in the AEE. Their mislocalisation in the stretched AEE cells may cause EC dysfunction through altered Ca2+ signalling thatcontributes to AF development.