Secretion of organic cations (OCs) across renal proximal tubules (RPTs) involves basolateral OCT2-mediated uptake from the blood, followed by apical MATE1/2-mediated efflux into the tubule filtrate. Whereas OCT2 supports electrogenic OC uniport, MATE is an OC/H+ exchanger. As assessed by epifluorescence microscopy, cultured CHO cells that stably express hMATE1 accumulated the fluorescent OC, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in hOCT2 expressing cells was largely restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of efflux of the prototypic OC, [3H]MPP, from MATE1-expressing CHO cells. Punctate accumulation of NBD-MTMA was markedly reduced by coexposure of MATE1-expressing cells with 5 μM bafilomycin (BAF), an inhibitor of the V-Type H-ATPase, and accumulation of [3H]MPP and [3H]NBD-MTMA was reduced by >30% by coexposure with 5 μM BAF. BAF had no effect on the initial rate of MATE1-mediated uptake of NBD-MTMA suggesting that the influence of BAF was a secondary effect involving inhibition of the V-type H-ATPase. The accumulation of [3H]MPP by isolated single non-perfused rabbit RPTs was also reduced >30% by coexposure to 5 μM BAF, suggesting that the native expression in RPTs of MATE protein within endosomes can increase steady-state OC accumulation. However, the rate of [3H]MPP secretion by isolated single perfused rabbit RPTs was not affected by 5µM BAF, suggesting that vesicles loaded with OCs+ are not likely to recycle into the apical plasma membrane at a rate sufficient to provide a parallel pathway for OC secretion.