Summary: Ventricular cells from adult rats were isolated enzymatically and used as a model system for determining what factors affect the release of adenosine triphosphate (ATP) from myocardial cells. The enzyme systems used to isolate cells were trypsin:collagenase; hya1uronidase:collagenase and dispase:collagenase. Adenosine triphosphate was released in greater amounts in response to hypoxia from cells freed by each of the enzymatic procedures. This occurred while the intracellular concentration of ATP remained constant. Experiments were then performed to determine whether the conditions that occur during myocardial ischaemia or hypoxia altered the release of ATP. Cells suspended in either oxygenated or anoxic buffer at a pH of 6.8 released a significantly lower amount of ATP than cells suspended in either condition at pH 7.4. To test the possibility that ATP was released from nucleotide-protein-Ca2+ complexes located in the sarcolemma, artificial disruption of these structures was carried out. Incubation of oxygenated cells with the chelating agent, ethyleneglycol-bis (B-aminoethyl ether)-N, N-tetraacetic acid (EGTA), stimulated the release of ATP in a hyperbolic relationship while incubation of anoxic cells with ethylenediamine tetraacetate (EDTA) stimulated the release of ATP in such a way that the pattern of release followed a sigmoid response with maximal amounts of ATP, 995±55 pmol·mg-2 protein, occurring in the presence of 0.1 to 2.0 mmol·litre-2 EDTA. By incubating cells with radioactive EDTA, there was no indication that EDTA entered the cells. No release of ATP above control levels occurred when EDTA was chelated with Ca2+ before being applied to isolated cells. These data suggest that the source of ATP found extracellularly may have been nucleotide-protein-Ca2+ complexes located in the sarcolemma, and further support the role of ATP as a coronary vasodilator during hypoxic conditions.