The interaction of the cationic spin probe 4-(N,N-dimethyl-N-dodecyl)-ammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl (Cat12) with intact mitochondria and submitochondrial particles was investigated as a function of salt concentration, pH and energization by ATP. In the presence of 1 mM Fe(CN)-36, which inhibits the probe reduction by the mitochondria, the probe signal is stable and shows both bound and free forms. The partition of the probe into mitochondrial membranes is decreased by various salts depending on the cation valency, indicating that the membrane is negatively charged (-10 to -15 mV at pH 7.0). The surface potential increases with pH from -3 mV at pH 5.0 to -18 mV at pH 8.0. Energization of intact mitochondria by ATP reduces the magnitude of both bound and free signals by more than 50%; the signal of the bound form slowly disappears on further incubation. The ATP effect is inhibited and also reversed by either oligomycin or CCCP. Similar effects of ATP were observed in mitoplasts but not in submitochondrial particles. In submitochondrial particles ATP has no effect on the probe signal or binding. These results suggest that the formation of membrane potential in mitochondria induces uptake and internal binding of the probe which results in broadening of the EPR signal of the internally bound probe. It is concluded that Cat12 is not a suitable probe for measurement of surface potential in energized mitochondria.