The major apurinic/apyrimidinic (AP) endonuclease of human cells, the Ape protein, incises DNA adjacent to abasic sites to initiate DNA repair and counteract the cytotoxic and mutagenic effects of AP sites. Here we address the determinants of Ape AP endonuclease activity using duplex DNA substrates that contain synthetic analogs of AP sites: tetrahydrofuranyl (F), propanediol (P), ethanediol (E), or 2-(aminobutyl)-1,3-propanediol (Q). The last of these, a branched abasic structure, was a poor substrate for which Ape had kcat > 1000-fold lower than for F. In contrast, the specificity constant (kcat/Km) for E or P of Ape purified from HeLa cells was only 5-8-fold lower than for F. Positioning a phosphorothioate ester immediately 5' to F inhibited Ape incision activity 20-fold (Rp isomer) or > 10,000-fold (Sp isomer). Although Ape did not have detectable endonuclease activity toward single-stranded substrates or unmodified double-stranded DNA, the enzyme displayed a low level of 3'-exonuclease activity for duplex DNA (< 0.03% of its AP endonuclease activity), which was influenced by the reaction conditions. The base positioned opposite F did not dramatically affect the cleavage efficiency of Ape, but an F:F arrangement was cleaved at approximately one-third of the efficiency of F:C. A 3'-mismatch diminished P and E cleavage only slightly and F not at all. A 5'-mismatch reduced the Ape cleavage rate 4-10-fold for F and approximately 100-fold for P and E. A series of substrates with F at different positions along the oligonucleotide showed that Ape requires > or = 4 base pairs 5' to the abasic site and > or = 3 base pairs on the 3'-side. The implications of these results for substrate recognition by Ape are discussed.