Saccharomyces cerevisiae Lpe10p is a homologue of the Mg 2+-channel-forming protein Mrs2p in the inner mitochondrial membrane. Deletion of MRS2, LPE10 or both results in a petite phenotype, which exhibits a respiratory growth defect on nonfermentable carbon sources. Only coexpression of MRS2 and LPE10 leads to full complementation of the mrs2lpe10 double disruption, indicating that these two proteins cannot substitute for each other. Here, we show that deletion of LPE10 results in a loss of rapid Mg2+ influx into mitochondria, as has been reported for MRS2 deletion. Additionally, we found a considerable loss of the mitochondrial membrane potential (Δψ) in the absence of Lpe10p, which was not detected in mrs2 cells. Addition of the K+H+-exchanger nigericin, which artificially increases Δψ, led to restoration of Mg2+ influx into mitochondria in lpe10Δ cells, but not in mrs2Δ/ lpe10Δ cells. Mutational analysis of Lpe10pΔ and domain swaps between Mrs2p and Lpe10p suggested that the maintenanceΔψ of and that of Mg2+ influx are functionally separated. Cross-linking and Blue native PAGE experiments indicated interaction of Lpe10p with the Mrs2p-containing channel complex. Using the patch clamp technique, we showed that Lpe10p was not able to mediate high-capacity Mg2+ influx into mitochondrial inner membrane vesicles without the presence of Mrs2p. Instead, coexpression of Lpe10p and Mrs2p yielded a unique, reduced conductance in comparison to that of Mrs2p channels. In summary, the data presented show that the interplay of Lpe10p and Mrs2p is of central significance for the transport of Mg2+ into mitochondria of S. cerevisiae.