In cardiomyocytes, cytosolic Ca removal is mediated by sarcoplasmic reticulum Ca ATPase (SERCA), sarcolemmal Na/Ca exchanger (NCX), plasmalemmal Ca ATPase and mitochondrial Ca uniporter (MCU). We quantified spatiotemporal inhomogeneities in cytosolic Ca removal in normal and diseased hearts and explored underlying mechanisms. Ca transients (1 Hz, Fluo-4 AM) were recorded using confocal microscopy in ventricular cardiomyocytes. Time constant of cytosolic [Ca] decay (tau_loc) was quantified at 1 μm intervals across the cell. In murine cardiomyocytes tau_loc distribution was inhomogeneous, with a maximal difference between cell regions of 237±9 ms and a variation coefficient (CV_tau) of 14±2% (mean±S.E.M., n=10 cells). tau_loc was not releated to the local amplitude or release kinetics of Ca. Coherent regions of fast (fastCaR, tau_loc < mean tau of whole cell) and slow (slowCaR) Ca removal had similar widths (4.6±0.2 vs. 5.1±0.5 im). Forskolin accelerated, and SERCA inhibitor cyclopiazonic acid (CPA) inhibited Ca removal significantly more in slowCaR than in fastCaR. In contrast, NCX-inhibitor SEA0400 slowed cytosolic Ca removal similarly in slowCaR and fastCaR. Also, CV_tau was unchanged in NCX+/- knock-out mice, suggesting no contribution of NCX to dyssynchrony in cytosolic Ca removal. tau_loc distribution was also inhomogenous in pig cardiomyocytes. Ca removal was more dyssynchronous in cardiomyocytes from chronic (4 weeks) ischemic pig myocardium (peri-infarct zone) vs. sham-operated pigs (CV_tau 24±1%vs. 20±1%, n=57 cells/group, p<0.05).Simultaneous recording of Ca transients and mitochondrial signal (mitotracker) unveiled a significant correlation between FastCaR regions and mitochondria (n=9 cells, p<0.05). Ru360 (MCU-inhibitor) slowed tau_loc more in FastCaR (to 126±12% vs. 112±10% of baseline in SlowCaR;p<0.05, n=5).Conclusion: Cytosolic Ca removal is dyssynchronous in mouse and pig cardiomyocytes. Ca removal in chronically ischemic cardiomyocytes is more dyssynchronous, suggesting a potential new mechanism of cardiomyocyte dysfunction. Mitochondria influence the differences in local Ca reuptake.