A cold/warm loading protocol was used to ester-load Rhod 2 into mitochondria and other organelles and Fluo 3 into the cytosol of adult rabbit cardiac myocytes for confocal fluorescence imaging. Transient increases in both cytosolic Fluo 3 and mitochondrial Rhod 2 fluorescence occurred after electrical stimulation. Ruthenium red, a blocker of the mitochondrial Ca²⁺ uniporter, inhibited mitochondrial Rhod 2 fluorescence transients but not cytosolic Fluo 3 transients. Thus the ruthenium red-sensitive mitochondrial Ca²⁺ uniporter catalyzes Ca²⁺ uptake during beat-to-beat transients of mitochondrial free Ca²⁺, which in turn may help match mitochondrial ATP production to myocardial ATP demand. After ester loading, substantial amounts of Ca²⁺-indicating fluorophores localized into an acidic lysosomal/endosomal compartment. This lysosomal fluorescence did not respond to electrical stimulation. Because fluorescence arose predominantly from lysosomes after the cold loading/warm incubation procedure, total cellular fluorescence failed to track beat-to-beat changes of mitochondrial fluorescence. Only three-dimensionally resolved confocal imaging distinguished the relatively weak mitochondrial signal from the bright lysosomal fluorescence.