HAP1, also known as APE/Ref-1, is the major apurinic/apyrimidinic (AP) endonuclease in human cells. Previous structural studies have suggested a possible role for the Asp-210 residue of HAP1 in the enzymatic function of this enzyme. Here, we demonstrate that substitution of Asp-210 by Asn or Ala eliminates the AP endonuclease activity of HAP1, while substitution by Glu reduces specific activity approximately 500-fold. Nevertheless, these mutant proteins still bind efficiently to oligonucleotides containing either AP sites or the chemically unrelated bulky p-benzoquinone (pBQ) derivatives of dC, dA and dG, all of which are substrates for HAP1. These results indicate that Asp-210 is required for catalysis, but not substrate recognition, consistent with enzyme kinetic data indicating that the HAP1-D210E protein has a 3000-fold reduced K(cat )for AP site cleavage, but an unchanged K(m). Through analysis of the binding of Asp-210 substitution mutants to oligonucleotides containing either an AP site or a pBQ adduct, we conclude that the absence of Asp-210 allows the formation of a stable HAP1-substrate complex that exists only transiently during the catalytic cycle of wild-type HAP1 protein. We interpret these data in the context of the structure of the HAP1 active site and the recently determined co-crystal structure of HAP1 bound to DNA substrates.