To study the relationship between stimulation frequency and intracellular Na+ concentration ([Na+]i), Langendorff-perfused rat hearts were loaded with the Na(+)-sensitive dye sodium-binding benzofuran isophthalate (SBFI). An intracellular calibration procedure allowed SBFI fluorescence to be transformed into [Na+]i. Compartmentation of SBFI was evaluated by permeabilizing sarcolemmal membranes with saponin and subcellular compartments with Triton X-100. Most of the indicator was located in the myoplasm (69%). When stimulation frequency was increased from 0 to 6 Hz, [Na+]i increased from 3.0 to 7.9 mM, whereas pressure amplitude (PA) declined to 49% of the maximum recorded at 2 Hz. Blocking sarcoplasmic reticulum (SR) Ca2+ uptake with 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ; 10 microM) at 2 Hz increased [Na+]i from 4.9 to 8.4 mM and decreased PA by 70%. Raising stimulation frequency then resulted in a further increase in [Na+]i and decline in PA. In conclusion, these data indicate that the rat heart is characterized by a negative pressure-frequency relationship associated with increasing [Na+]i at higher heart rates. After inhibition of SR Ca2+ uptake, [Na+]i further increases, whereas PA declines with increasing stimulus frequency. It is suggested that part of the rise of [Na+]i with stimulus frequency and TBQ may be associated with increased Ca2+ extrusion and Na+ influx on the Na+/Ca2+ exchange system.