It is known that the level of ATP in the interstitial spaces within the heart during ischemia or hypoxia is elevated due to its release from a number of cell types, including cardiomyocytes. However, the mechanism by which ATP is released from these myocytes is not known. In this study, we examined a possible involvement of the ATP-conductive maxi-anion channel in ATP release from neonatal rat cardiomyocytes in primary culture upon ischemic, hypoxic or hypotonic stimulation. Using a luciferin-luciferase assay, it was found that ATP was released to the bulk solution when the cells were subjected to chemical ischemia, hypoxia or hypotonic stress. The swelling-induced ATP release was inhibited by carboxylate- and stilbene-derivative anion channel blockers, arachidonic acid and Gd³⁺, but not by glibenclamide. The local concentration of ATP released near the cell surface of a single cardiomyocyte, measured by a biosensor technique, was found to exceed the micromolar level. Patch-clamp studies showed that ischemia, hypoxia or hypotonic stimulation induced the activation of single-channel events with a large unitary conductance (~390 pS). The channel was selective to anions and showed significant permeability to ATP^4- (P_ATP/P_Cl ~ 0.1) and MgATP^2- (P_ATP/P_Cl ~ 0.16). The channel activity exhibited pharmacological properties essentially identical to those of ATP release. These results indicate that neonatal rat cardiomyocytes respond to ischemia, hypoxia or hypotonic stimulation with ATP release via maxi-anion channels.