1. Adult rat heart cells were isolated enzymically and ATP was identified in the cell suspension using the firefly luminescence technique. Adenosine 5'-triphosphate (ATP) was not detected from cell suspensions obtained from hearts which had been left asystolic for 10 min.2. It was found that ATP 0.34 +/- 0.22 muM/mg protein was released by cells kept in an oxygenated condition, while ATP 1.28 +/- 0.41 muM/mg protein was initially released by cells made hypoxic.3. Addition of Ca(2+) in a concentration of 2 mM caused cells to initially extrude ATP 0.40 +/- 0.14 muM/mg protein. This was attributed to an inotropic effect.4. Extracellular ATPase activity in the fluid suspension was partially characterized, giving a K(m) of 13 muM and a V/2 of hydrolysed ATP 18.3 muM/min at 37 degrees C. Q(10) was found to be 4 between 25 and 37 degrees C. Enzyme activity remained unaffected by either hypoxic conditions or ouabain.5. If these amounts of ATP are released from myocardial cells rendered hypoxic in vivo, then it must be concluded that ATP plays a principal role in the local control of myocardial blood flow.6. It is proposed that release of ATP occurs through the sarcolemma from an intracellular pool, and that alteration of the configuration of structural membrane protein controls the amounts of ATP extruded.