Fluorescence, absorbance, and binding of a mitochondrial membrane potential-sensitive probe, rhodamine 800 (rhod800), were measured in isolated rat mitochondria, hepatocytes, cardiomyocytes, and hearts in the presence or absence of mitochondrial uncouplers. Excitation of rhod800 was achieved with laser diodes (690 or 670 nm) and resulted in a fluorescence peak at 720 nm. Greater than 99% of rhod800 (1 µM) was taken up from the buffer by energized mitochondria. This resulted in a fluorescence decrease by 77% (13% in de-energized mitochondria). Sixty-seven percent of rhod800 was taken up by cardiomyocytes and 75% by hepatocytes resulting in the fluorescence decrease by 16% and 37%, respectively, which were reversed by approximately 10% upon cell uncoupling. In hearts, binding, absorbance, and fluorescence were almost uncoupler-insensitive possibly due to rhod800 interaction outside of mitochondria. Fluorescence of the hearts perfused with 27.5 and 55 nM rhod800 was measured in orthogonal and reflection modes. The former provided deep tissue penetration (approximately a centimeter); however, nonlinearity between absorbance and fluorescence was evident. In the latter setting, depth of tissue penetration was approximately a millimeter, which eliminated an inner filter effect and restored linearity. We concluded that excessive hydrophobicity of rhod800 complicates detection of energy-dependent fluorescence changes in myocardium.