In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red-120 agarose, from which it was eluted by ATP, less effectively by ADP and AMP but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC. [alpha-32P]BzATP bound to purified VDAC at two or more binding sites with apparent high- and low-binding affinities. MALDI-TOF analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC-BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively-charged nucleotide species. VDAC treatment with the lysyl residue-modifying reagent, fluorescein 5'-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide binding sites were localized using chemical and enzymatic cleavage. Digestion of [-32P]BzATP-labeled VDAC with CNBr or with V8 protease resulted in the appearance of ~17 and ~14 kDa labeled fragments. Further digestion, HPLC separation and sequencing of selected V8-resulted peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N- and C-termini of VDAC. Thus, the presence of two or more nucleotide binding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.