Purinergic signalling plays a crucial role in the physiology of the heart. In the present study, we examined the involvement of maxi-anion channels in ATP release from primary cultures of cardiomyocytes of humanely killed neonatal rats and their distribution pattern on the sarcolemmal surface. Using a luciferin-luciferase assay, it was found that ATP was released to the bulk solution when the cells were subjected to chemical ischaemia, hypoxia or hypotonic stress. The cell surface ATP level of a single cardiomyocyte, measured by a biosensor technique, was found to exceed a micromolar level. Conventional patch-clamp studies showed that all three stimulation induced the activation of single-channel events with a large unitary conductance (390 pS). The pharmacological properties of the swelling-induced ATP release and maxi-anion channel were similar. 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). After taking a 3-D image of the cell by a scanning ion conductance microscopy technique in which a fine-tipped patch pipette was employed as a probe to monitor the cell surface, the same pipette was employed for patching the specified regions on the cells. We found that the density of ATP-conductive maxi-anion channel was higher in the central region of the cell body compared to the cell extensions. These results indicate that maxi-anion channels are distributed on the surface of sarcolemma predominantly near the cell body centre and serve as a pathway for ATP release in hypotonic, hypoxic and ischemic conditions.