We investigated the use of rhodamine 123 (R123), tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE) as fluorescent probes to monitor the membrane potential of mitochondria. These indicator dyes are lipophilic cations accumulated by mitochondria in proportion to DeltaPsi. Upon accumulation, all three dyes exhibit a red shift in both their absorption and fluorescence emission spectra. The fluorescence intensity is quenched when the dyes are accumulated by mitochondria. These properties have been used to develop a method to dynamically monitor DeltaPsi of isolated rat heart mitochondria using a ratio fluorescence approach. All three dyes bound to the inner and outer aspects of the inner mitochondrial membrane and, as a result, were accumulated by mitochondria in a greater quantity than predicted by the Nernst equation. Binding to mitochondria was temperature-dependent and the degree of binding was in the order of TMRE > R123 > TMRM. The internal and external partition coefficients for binding were determined to correct for binding in the calculation of DeltaPsi. All three dyes suppressed mitochondrial respiratory control to some extent. Inhibition of respiration was greatest with TMRE, followed by R123 and TMRM. When used at low concentrations, TMRM did not suppress respiration. The use of these dyes and ratio fluorescence techniques affords a simple method for measurement of DeltaPsi of isolated mitochondria. We also applied this approach to the isolated perfused heart to determine whether DeltaPsi could be monitored in an intact tissue. Wavelength scanning of the surface fluorescence of the heart under various conditions after accumulation of TMRM indicated that the mitochondrial matrix-induced wavelength shift of TMRM also occurs in the heart cytosol, eliminating the use of this approach in the intact heart.