The absorption or fluorescence spectrum of rhodamine 6G upon interaction with mitochondria was changed following the addition of substrate. A similar spectral change was obtained for the binding of positively charged rhodamine 6G to negatively charged chemicals such as sodium dodecyl sulfate, dextran sulfate, pentachlorophenol, and tetraphenylboron. This indicates that the positively charged dye molecules become exposed to the negatively charged region on the mitochondrial membrane generated by the addition of substrate, which may be closely associated with the charge separation of mitochondria.