The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo) is a major factor in the maintenance of the integrity of the human genome. There are estimates that this enzyme is responsible for eliminating as many as 10(5) potentially mutagenic and genotoxic lesions from the genome of each cell every day. Furthermore, inhibition of AP endonuclease may be effective in decreasing the dose requirements of chemotherapeutics used in the treatment of cancer as well as other diseases. Therefore, it is essential to accurately and directly characterize the enzymatic mechanism of AP endo. Here we describe specifically designed double-stranded DNA oligomers containing tetrahydrofuran (THF) with a 5'-phosphorothioate linkage as the abasic site substrate. Using H(2)(18)O during the cleavage reaction and leveraging the stereochemical preferences of AP endo and T4 DNA ligase for phosphorothioate substrates, we show that AP endo acts by a one-step associative phosphoryl transfer mechanism on a THF-containing substrate.