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ABSTRACT.Derivative spectrophotometry was used to study the interaction of the cationic uncoupler triS-C4 (5) with mitochondria.The uncoupling action of this dye is dependent on the presence of Pi in the incubation medium.The second derivative spectrum of the dye changed with the incubation period, becoming similar to the spectrum in chloroform; but, after a time, the spectral pattern reverted to the original spectrum. The change in the spectrum in the presence of Pi was much more rapid than in its absence. The degree of spectral change agreed with the relative amount of bound dye determined directly. Thus, the spectral change reflects the binding of dye to the mitochondria, dependent on their energy state. The greater binding without Pi does not cause uncoupling but does cause shrinkage. In contrast, the lesser binding in the presence of Pi causes uncoupling and the swelling of mitochondria.These facts indicate that the dye does not penetrate the mitochondrial membrane. This refutes the idea that uncoupling by lipophilic cations is caused by the electrophoretic transfer of the uncoupler to the mitochondrial matrix space.