Human apurinic/apyrimidinic (AP) endonuclease APE1 catalyses the hydrolysis of phosphodiester bonds on the 5 side of an AP-site (in the base excision repair pathway) and of some damaged nucleotides (in the nucleotide incision repair pathway). The range of substrate specificity includes structurally unrelated damaged nucleotides. Here, to examine the mechanism of broad substrate specificity of APE1, we performed pulsed electron–electron double resonance (PELDOR) spectroscopy and pre-steady-state kinetic analysis with Förster resonance energy transfer (FRET) detection of DNA conformational changes during DNA binding and lesion recognition. Equilibrium PELDOR and kinetic FRET data revealed that DNA binding by APE1 leads to noticeable damage-dependent bending of a DNA duplex. Molecular dynamics simulations showed that the damaged nucleotide is everted from the DNA helix and placed into the enzyme’s binding pocket, which is formed by Asn-174, Asn-212, Asn-229, Ala-230, Phe-266 and Trp-280. Nevertheless, no damage-specific contacts were detected between these amino acid residues in the active site of the enzyme and model damaged substrates containing 1,N6-ethenoadenosine, -adenosine, 5,6-dihydrouridine or F-site. These data suggest that the substrate specificity of APE1 is controlled by the ability of a damaged nucleotide to flip out from the DNA duplex in response to an enzyme-induced DNA distortion.