The mitochondrial outer membrane contains two protein translocators: the TOM40 and TOB/SAM complexes. Mdm10 is distributed in the TOB complex for beta-barrel protein assembly and in the MMM1 complex for tethering of the endoplasmic reticulum and mitochondria. Here, we establish a system in which the Mdm10 level in the TOB complex--but not in the MMM1 complex--is altered to analyse its part in beta-barrel protein assembly. A decrease in the Mdm10 level results in accumulation of in vitro imported Tom40, which is a beta-barrel protein, at the level of the TOB complex. An increase in the Mdm10 level inhibits association not only of Tom40 but also of other beta-barrel proteins with the TOB complex. These results show that Mdm10 regulates the timing of release of unassembled Tom40 from the TOB complex, to facilitate its coordinated assembly into the TOM40 complex.