The endonuclease (AP endonuclease) specific for apurinic/apyrimidinic sites (AP sites) has been purified and characterized from normal human autopsy liver. Three chromatographically distinct species of AP endonuclease were resolved by phosphocellulose chromatography. The species had molecular weights ranging from 24,000 to 31,000. Species 3 was significantly more thermostable than was either species 1 or species 2. Species 2 had an apparent Km of 400 nM AP sites whereas species 3 had an apparent Km of 2000 nM AP sites. All of the species required Mg2+ for activity. The concentration of Mg2+ which resulted in half maximal velocity was 5 mM for species 3 but was only 1.2 mM for species 1. The observed differences in physical and catalytic properties of the three species suggest they are isozymes. Incision of damaged DNA by the three putative isozymes was restricted to AP sites and resulted in the production of 3'hydroxyl and 5' phosphorylated termini.