Spatial heterogeneity of action potential properties has been related to cardiac arrhythmogenesis. In this study we used laser scanning confocal microscopy in conjunction with the fast potentiometric dye ANNINE-6 to monitor changes in cardiomyocyte transmembrane potentials in Langendorff-perfused mouse hearts on a subcellular scale. Line-scan images from up to three neighboring cardiomyocytes were obtained during continuous electrical stimulation at 3 Hz. Fluorescence changes for each cardiomyocyte along the scan line were resolved from the corresponding line-scan image. Peak changes in fluorescence intensity during an action potential exceeded 20%. Signal-to-noise ratio of the optical signal was >20. Action potential durations were not significantly different between adjacent cardio- myocytes under our conditions. We conclude that this imaging technique can be used to investigate cell-to-cell repolarization heterogeneity in the intact heart.