By using a band mobility shift assay, deoxyinosine 3'-endonuclease, an Escherichia coli enzyme which recognizes deoxyinosine, AP site, urea residue, and base mismatches in DNA, was shown to bind tightly to deoxyinosine-containing oligonucleotide duplexes. Two distinct protein-DNA complexes were observed, the faster migrating complex (complex I, Kd = 4 x 10(-9) M) contained one molecule of deoxyinosine 3'-endonuclease, while the slower migrating complex (complex II, Kd = 4 x 10(-7) M) contained two molecules of the protein bound to every molecule of duplex DNA. The endonucleolytic activity of deoxyinosine 3'-endonuclease paralleled the formation of the complex I. Interestingly, deoxyinosine 3'-endonuclease exhibited similar affinities for both the substrate and the nicked duplex product and thus remained bound to the DNA after the cleavage reaction. The formation of a stable complex required the presence of a duplex structure 5' to the deoxyinosine residue. DNase I footprinting revealed that deoxyinosine 3'-endonuclease protected 4-5 nucleotides 5' to the deoxyinosine, and when complex II was formed, at least 13 nucleotides 3' to deoxyinosine were protected. Based on these results, a model is proposed for the interaction of deoxyinosine 3'-endonuclease with DNA containing deoxyinosine.