We characterized the Na(+)-sensitive dye benzofuran isophthalate (SBFI) with fluorescence microscopy in isolated, adult rat ventricular myocytes. When cells were loaded with SBFI by incubation with 10 microM of the acetoxymethyl ester, fluorescence excitation spectra were markedly attenuated below 340 nm and the isoemissive point was blue shifted by approximately 25 nm when compared with spectra from SBFI acid in buffered solutions. Fluorescence intensity (49 +/- 3%) was partially released by permeabilization of the sarcolemma with digitonin, suggesting that one-half of the dye molecules are sequestered subcellularly in a compartment shown most likely to be mitochondrial. Intracellular Na+ concentration ([Na+]i) was determined by in situ calibration using cation-selective ionophores and was found to be 14 +/- 2 mM in cells studied at 37 degrees C. The relative importance of Na+ efflux mechanisms in myocytes was investigated. Substitution of Ca2+ and Mg2+ with EGTA in the superfusing medium resulted in a reversible rise of [Na+]i from 13 +/- 2 to 31 +/- 5 mM, which was blocked by 1 microM verapamil. Cellular efflux of Na+ after loading in this manner was found to be insensitive to blockade of Na(+)-Ca2+ exchange but was abolished when Na(+)-K(+)-ATPase was inhibited with zero extracellular K+ concentration. We conclude that SBFI can be used to measure [Na+]i in ventricular cells nondestructively and without impalement. Na+ efflux after loading by Ca2+ and Mg2+ withdrawal is mediated by the Na+ pump with no measurable contribution from Na(+)-Ca2+ exchange.