Cardiac Ca signaling is organized into structurally and functionally specialized compartments that include junctional Ca release units (CRUs) coupled to L-type Ca channels (LTCC) and LTCC-free non-junctional CRUs. Little is known about subcellular diferences in patholgic Ca handling and their role in cardiac arrhythmogenesis. We have shown that diminished Ca signaling refractoriness in diseased myocytes contributes to their susceptability for diastolic Ca waves (DCWs). The objective of present study was to define the subcellular determinants of arrhythmogenic DCWs by quantifying functional differences in Ca signaling from anatomically distinct sites (junctional vs. non-junctional) and their relative roles in the genesis of DCWs. We employed high resolution 2D confocal Ca imaging in ventricular myocytes isolated from normal and post-myocardial infarction (MI) arrhythmia-prone canine hearts. Ca release sites were categorized as early- or delayed-response (corresponding to junctional and non-junctional regions, respectively) based on activation time following electrical stimulation. MI (but not control) myocytes exhibited regular DCWs when paced in the presence of isoproterenol. These DCWs predominantly originated from early-response sites. Consistent with this observation, majority of spontaneous Ca sparks also occurred at early-response sites. Interestingly, after stimulated Ca release the restitution time of Ca sparks was similar between control and MI myocytes. Furthermore, application of a two-pulse restitution protocol revealed similar rates of Ca release recovery between control and MI myocytes at the early-response sites. However, the Ca release recovery at late-response sites was markedly abbreviated in MI cells. These results suggest that increased propensity of MI myocytes toward arrhythmogenic DCWs can be attributed to diminished refractoriness of non-junctional CRUs facilitating the transition of Ca sparks at eager junctional sites to self-propagating Ca waves via the recruitment of non-junctional sites.