The composition and pH of atrial-specific granules of rat heart were determined by electron probe x-ray microanalysis and fluorescence microscopy, respectively. The high (75 mmol/kg of dry weight) calcium content and higher than cytoplasmic concentration of chloride in atrial-specific granules were visualized in high-resolution x-ray maps. The Cl- content of granules and cytoplasm decreased and a bromide gradient (granule greater than cytoplasm) was established during incubation in low-chloride, NaBr-containing solutions. Scanning confocal fluorescence light microscopy of live atria incubated with acridine orange demonstrated dye accumulation, indicative of low intragranular pH. We conclude that the granules represent a hitherto unrecognized intracellular store of cardiac calcium and can develop and maintain an anion gradient, presumably through cotransport by means of a proton-pumping ATPase.