Mammalian multidrug and toxic compound extrusion (MATE) proteins are classified into three subfamilies, classes I, II and III. Two of these families have been shown to act as polyspecific H⁺-coupled transporters of organic cations (OCs) at final excretion steps in liver and kidney (Otsuka M et al., Proc. Natl. Acad. Sci. USA, 102: 17923-17928, 2005; Omote H et al., Trends Pharmacol. Sci. 27: 587-593, 2006). Rodent MATE2 proteins belong to the third class of MATE transporters whose molecular nature as well as transport properties remain to be characterized. In the present study, we investigated the transport properties and localization of mouse MATE2 (mMATE2). Upon expression in HEK293 cells, mMATE2 localized to the intracellular organelles and the plasma membrane. Mouse MATE2 mediated pH-dependent tetraethylammonium (TEA) transport with a substrate specificity similar to but distinct from that of mMATE1, which prefers N-methyl nicotinamide (NMN) and guanidine as substrates. Mouse MATE2 expressed in insect cells was solubilized and reconstituted with bacterial H⁺-ATPase into liposomes. The resultant proteoliposomes exhibited ATP-dependent uptake of TEA that was sensitive to carbonyl cyanide 3-chlorophenylhydrazone (CCCP) but unaffected by valinomycin in the presence of K⁺. Immunological techniques using specific antibodies revealed that mMATE2 was specifically expressed in testicular Leydig cells. Thus, mMATE2 appears to act as a polyspecific H⁺/OC exporter in Leydig cells. It is concluded that all classes of mammalian MATE proteins act as polyspecific and electroneutral transporter of organic cations.