The trinuclear cyanine dye, tri-S-C7(5), at about 10 microM stimulated State 4 respiration of rat liver mitochondria more than 6-fold and released oligomycin-inhibited respiration completely. Thus, the dye is concluded to be a very effective cationic uncoupler of oxidative phosphorylation in mitochondria. However, for exhibition of its uncoupling action, the presence of Pi (or arsenate) was necessary, and a phosphate-transport inhibitor, N-ethylmaleimide or mersalyl, inhibited its action. The stimulation of phosphate transport via the Pi carrier by the dye is suggested to be directly related to the uncoupling action.