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, K = 4 × 10M) contained one molecule of deoxyinosine 3′-endonuclease, while the slower migrating complex (complex II, K = 4 × 10M) 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.