A 3'-5' exonuclease that excises the nucleotide analogs ara-CMP and F-ara-AMP incorporated at 3'-ends of DNA was purified from the nuclei of: 1) primary human chronic lymphocytic leukemia cells; 2) primary and established human acute myeloblastic leukemia cells; 3) lymphocytes obtained from healthy individuals. The activity of this nuclear exonuclease (exoN) is elevated approximately 6-fold in ara-C-resistant leukemia cells as compared to drug-sensitive cells and it differs between two healthy individuals and among three leukemia patients. ExoN is a 46 kDa monomer, requires 50 mM KCl and 1 mM magnesium for optimal activity, and shows a preference for single-stranded over duplex DNA. Its physical and enzymatic properties indicate that exoN is a previously uncharacterized enzyme whose activity may confer resistance to clinical nucleoside analogs in leukemia cells.