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Ventricular myocytes remodel after myocardial infarction (MI). Myocytes hypertrophy, mitochondria are affected and the density of T-tubules reduces, while their distance to ryanodine receptors (RyRs) and mitochondria increases. Reactive oxygen species (ROS) production may increase and modulate RyR function within microdomains. We investigated the hypothesis that distance to mitochondria may be an independent determinant of RyR activity, in particular after MI.We studied permeabilized myocytes, where the influences of sarcolemma, bulk [Ca] and [ROS] in sham vs. MI are minimized. Cells were isolated from 6-weeks post-MI pig hearts, from the area adjacent to the MI and matching tissue in sham. xyt scanning served to characterize Ca sparks at the focal plane, relative to the xyz distribution of mitochondria. Mitochondrial z-stacks were processed using standard procedures, while a xyt spark detector sharing features with published algorithms [Kong et al (2008) Biophys J. 95(12):6016-24; Banyasz et al. (2007) Biophys J. 92(12):4458-65] was created. Spark frequency was plotted as a function of the distance between each spark's center of mass (at first detection) and its closest outer mitochondrial membrane (OMM).Control experiments in healthy permeabilized cells showed that, in our conditions, ROS were produced by mitochondria. In healthy and diseased myocytes, most sparks occurred within 0.5 microns of an OMM. However, it was found that the relationship between spark frequency and spark center-OMM distance did not change with MI. Yet in experiments in intact cells, scavenging of mitochondrial ROS affected spark production in MI predominantly in RyRs at a distance from the T-tubules. These results might indicate that ROS production by mitochondria likely affects all RyRs due to their ubiquitous nature but that further regulatory factors of RyR activity may be related to the presence of an intact sarcolemma.