AP endonuclease cleaves the phosphodiester bond 5'- to the AP (apurinic or apyrimidinic) sites and is one of the major enzymes involved in base excision repair. So far, the properties of several archaeal AP endonuclease homologues have been characterized in vitro, but little is known about their functions in vivo. Herein, we report on the biochemical and genetic analysis of two AP endonucleases, SisExoIII and SisEndoIV, from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A. Both SisExoIII and SisEndoIV exhibit AP endonuclease activity, but neither of them has 3'-5' exonuclease activity. SisExoIII and SisEndoIV have similar K (M) values on the substrate containing an AP site, but the latter cleaves the AP substrate at a dramatically higher catalytic rate than the former. Unlike other AP endonucleases identified in archaea, SisExoIII and SisEndoIV do not exhibit any cleavage activity on DNA having oxidative damage (8-oxo-dG) or uracil. Genetic analysis revealed that neither gene is essential for cell viability, and the growth of a dagger SiRe_2666 (endoIV), a dagger SiRe_0100 (exoIII), and a dagger SiRe_0100a dagger SiRe_2666 is not affected under normal growth conditions. However, a dagger SiRe_2666 exhibits higher sensitivity to the alkylating agent methyl methanesulfonate (MMS) than a dagger SiRe_0100. Over-expression of SiRe_0100 can partially complement the sensitivity of a dagger SiRe_2666 to MMS, suggesting a backup role of SisExoIII in AP site processing in vivo. Intriguingly, over-expression of SisEndoIV renders the strain more sensitive to MMS than the control. Taken together, we conclude that SisEndoIV, but not SisExoIII, is the main AP endonuclease that participates directly in base excision repair in S. islandicus.