The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane is a small abundant protein found in all eukaryotic kingdoms which forms a voltage-gated pore when incorporated into planar lipid bilayers. VDAC is also the site of binding of the metabolic enzymes hexokinase and glycerol kinase to the mitochondrion in what may be a significant metabolic regulatory interaction. Recently, there has been speculation that there may be multiple forms of VDAC in mammals which differ in their localization in the outer mitochondrial membrane and in their physiological function. In this report, we describe the identification and characterization of two human cDNAs encoding VDAC homologs (HVDAC1 and HVDAC2). To confirm VDAC function, each human protein has been expressed in yeast lacking the endogenous VDAC gene. Human proteins isolated from yeast mitochondria formed channels with the characteristics expected of VDAC when incorporated into planar lipid bilayers. In addition, expression of the human proteins in such strains can complement phenotypic defects associated with elimination of the endogenous yeast VDAC gene. Since VDAC is the site of binding of hexokinase to the outer mitochondrial membrane, the binding capacity of each VDAC isoform expressed in yeast mitochondria was assessed. When compared with the binding of hexokinase to mitochondria lacking VDAC, the results show that mitochondria expressing HVDAC1 are capable of specifically binding hexokinase, whereas mitochondria expressing HVDAC2 only bind hexokinase at background levels. The expression of each human cDNA has been assessed by Northern blot and polymerase chain reaction techniques. With one exception, each is expressed in all human cell lines and tissues examined.