Human apurinic/apyrimidinic endonuclease (APE1) is an enzyme of DNA base excision repair (BER) which catalyzes endonucleolytic cleavage immediately 5' to abasic (AP) sites. APE1 has long been thought to act on AP sites only in double stranded (ds) DNA, in order to generate the appropriate site for insertion of the correct nucleotide of DNA repair synthesis effected by DNA polymerase beta. We now present evidence that APE1 also acts on AP sites in single-stranded (ss) DNA. The catalytic efficiency of this activity (defined within as k(cat)/Km) is approximately 20-fold less than the activity against AP sites in ds DNA, with the disparity stemming largely from a difference in Km. Similar to its action on AP sites in ds DNA, catalysis of endonucleolytic cleavage of ss DNA by APE1 is Mg(2+) dependent, DNA N-glycosylase independent, and requires an active site aspartate. In contrast to its activity against AP sites in ds DNA, APE1 does not display product inhibition when acting on an AP site in ss DNA. We suggest that this novel activity is related to the processing of DNA N-glycosylase initiated BER in ss DNA perhaps during replication and/or transcription.