Newly synthesized precursors are transported into mitochondria through an outer membrane translocase, TOM. Tom40, a central pore-forming component, interacts directly with precursors to help them translocate across the outer membrane. We identified a new isoform of rat Tom40, Tom40B, which is conserved among mammals and exhibits significant similarities to Tom40 in other eukaryotes. Tom40B is an integral protein localized on the mitochondrial outer membrane, and expressed widely in all tissues examined except testis. Deletion mutant analysis revealed that the 28 amino acid residues at the carboxyl terminus were crucial for the mitochondrial targeting of Tom40B. Tom40B co-precipitated with other Tom components and formed a large protein complex. Furthermore, Tom40B directly bound to precursors of the matrix-targeted proteins with high affinities, comparable to those of Tom40A, a previously identified isoform. These findings indicate that Tom40B is a functional component of mitochondrial outer membrane translocase.