We have previously reported that the 3,N4-benzetheno-dC (p-BQ-dC) endonuclease activity found in HeLa cells is a novel function of the major human AP endonuclease (HAP1) [Hang et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 13737-13741]. In this study, we compare the enzymatic and biochemical properties of the enzyme toward p-BQ-dC and an AP site in a defined oligonucleotide. A comparative analysis of the specificity constants (Kcat./Km) for p-BQ-dC and an AP site indicates that the AP site is the preferred substrate. The enzyme does not cleave other structurally related exocyclic adducts and modified nucleosides such as 1,N6-etheno-dA, 3,N4-etheno-dC, 1, N2-etheno-dG, 1,N2-propano-dG, 8-oxo-dG, and thymine glycol. The p-BQ-dC activity requires a double-stranded DNA substrate and is affected by the base in the opposite strand, with maximal activity for a p-BQ-dC.G pair and minimal activity for a p-BQ-dC.C pair. The p-BQ-dC activity also requires Mg2+, Mn2+, or Zn2+ with optimal concentration spectra similar to those for the AP function. The optimal pH ranges for these two functions are also similar to each other (5.5-6.5). Six mutant HAP1 proteins containing single amino acid substitutions were assayed in parallel for comparison of their activities toward p-BQ-dC and the AP site. These mutants either concomitantly lost (N212A, D210N) or had reduced (D219A, E96A, and N212Q) or unchanged (H116N) p-BQ-dC and AP activities. This parallelism strongly supports the hypothesis that cleavage of p-BQ-dC requires the same catalytic active site as that proposed for the AP function. This dual activity toward two structurally unrelated substrates, an AP site and a bulky exocyclic adduct, has implications for substrate recognition. The AP site and p-BQ-dC cause different changes in the local conformation around the lesion as it is visualized by molecular modeling.