An ion-selective electrode specific for the 1-methyl-4-phenylpyridinium ion (MPP+) was developed which allowed the measurement of the initial rate of uptake and the extent of accumulation of MPP+ by rat liver mitochondria. Using this electrode we demonstrated that the initial rate of uptake of MPP+ was not saturable and that the distribution of MPP+ across the mitochondrial inner membrane did not equilibrate with the membrane potential, in contrast to other lipophilic cations such as the methyltriphenyl phosphonium ion (TPMP+). Furthermore, incubation of mitochondria respiring on succinate with MPP+ decreases the membrane potential. The possibility that the interaction of MPP+ with mitochondria may be more complex than accumulation followed by inhibition of site 1, suggested by these and other data, is discussed.