Voltage-dependent anion channels (VDACs) are small pore-forming channels found in the outer membrane of mitochondria. VDACs translocate adenine nucleotides and are the binding sites for several cytosolic kinases important in intermediary metabolism. Recently two human VDAC cDNAs (HVDAC1 and HVDAC2) were isolated, and possible orthologues of these genes have been isolated from mouse, bovine, and rat tissues. We report the isolation of a novel third VDAC cDNA from the mouse, designated MVDAC3. The deduced MVDAC3 protein is approximately 70% identical to the previously isolated MVDAC1 and MVDAC2 proteins. The MVDAC3 gene was mapped by an interspecies backcross panel to mouse chromosome 8. A database search using the mouse VDACs identified a second yeast VDAC-like gene that retains about 20% amino acid sequence identity with the mouse VDAC genes and 50% identity with the previously isolated yeast VDAC gene. The phylogenetic relationship of the eukaryotic VDAC genes is presented.