Apurinic/apyrimidinic (AP) endonuclease (Ape1) is the major cellular enzyme responsible for repairing AP-sites in DNA. It can cleave the DNA phosphodiester backbone immediately 5' to an AP-site. Ape1 also shows 3'-phosphodiesterase activity, a 3'-phosphatase activity, and an RNaseH activity. However, regarding its exonuclease activity, it remains controversial whether human Ape1 may possess a 3'-5' exonuclease activity. During the Course of study to search for the major nuclease activity to double-stranded DNA in human leukemia cells, we purified a 37 kDa Mg2+-dependent exonuclease from cytosolic fraction of human leukemia U937 cells. Surprisingly, this exonuclease is Ape1. We demonstrated for the first time that Ape1 possesses a significant activity as major 3'-5' exonuclease in human leukemia cells. In addition, we also observed that translocation of cytoplasmic Ape1 into nucleus occurs during DNA damage. Published by Elsevier Inc.