ATP is released by numerous cell types in response to mechanical strain. It then acts as a paracrine or autocrine signalling molecule, inducing a variety of biological responses. In this work we addressed the question whether mechanical force acting on the membranes of contracting cardiomyocytes during periodical longitudinal shortening can stimulate the release of ATP. Electrically stimulated isolated adult rat cardiomyocytes as well as spontaneously contracting mouse cardiomyocytes derived from embryonic stem cells were assayed for ATP release using luciferase and a sensitive CCD-camera. Sensitivity of soluble luciferase in the supernatant of cardiomyocytes was 100 nM ATP, which is about 10-fold below the EC50 values for most purinergic receptors expressed in the heart (1.5-20µM). Light intensities were not different over resting or contracting adult rat cardiomyocytes. Similar results were obtained with ES-cell-derived contracting mouse cardiomyocytes. ATP release was only measurable from obviously damaged or permeabilised cells. To increase selectivity and sensitivity of ATP detection we have targeted a recombinant luciferase to the sarcolemmal membrane using a WGA-IgG-linker. Contraction of labelled adult rat cardiomyocytes was not associated with measurable bioluminescence. However, when endothelial cells (HUVEC) were targeted with membrane-bound luciferase, shear stress-induced ATP release could be clearly detected, demonstrating the sensitivity of the detection method. In the present study we did not detect ATP release from contracting cardiomyocytes on the single cell level despite of adequate sensitivity of the detection system. Thus, deformation of the contracting cardiomyocyte is not a key stimulus for the release of cellular ATP.