The spatial distribution of intracellular Ca2+ concentration was determined by fluorescent digital imaging microscopy in fura-2-loaded quiescent cardiac myocytes isolated from guinea pig ventricle. Fluorescent ratio images revealed discrete as well as clustered bright fluorescent spots ("hot spots"), which occupied approximately 20-50% of an individual cell's area. The fluorescent intensity and the area of the hot spots were increased by agents that deplete Ca2+ in the sarcoplasmic reticulum, namely, ryanodine (20-40 nM) and caffeine (5-15 mM). However, when cells were exposed to agents that deplete mitochondrial Ca2+, such as the protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP, 100-300 nM), or the inhibitor of electron transport, antimycin A (4-40 nM), the fluorescent intensity and the area of the hot spots were reduced. These results indicate that the spatial distribution of intracellular Ca2+ concentration in the ventricular myocytes of guinea pig is quite heterogeneous. The ability of CCCP and antimycin A, but not of caffeine and ryanodine, to reduce the fluorescent intensity in the hot spots implies that Ca2+ compartmentation in the mitochondria is largely responsible for the intracellular Ca2+ heterogeneity seen in the present study.