Escherichia coli endonuclease VIII (endo VIII) was identified as an enzyme that, like endonuclease III (endo III), removes radiolysis products of thymine including thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, and urea from double-stranded plasmid or phage DNA and cleaves the DNA strand at abasic (AP) sites (Melamede, R. J., Hatahet, Z., Kow, Y. W., Ide., H., and Wallace, S. S. (1994) Biochemistry 33, 1255-1264). Using apparently homogeneous endo VIII protein, we now show that endo VIII removes from double-stranded oligodeoxyribonucleotides the stable oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil. Endo VIII cleaved the damage-containing DNA strand by beta,delta-elimination as does formamidopyrimidine DNA glycosylase (Fpg). Like Fpg, endo VIII also excised the 5'-terminal deoxyribose phosphate from an endonuclease IV (endo IV) pre-incised AP site. Thus, in addition to amino acid sequence homology (Jiang, D., Hatahet, Z., Blaisdell, J., Melamede, R. J., and Wallace, S. S. (1997) J. Bacteriol. 179, 3773-3782), endo VIII shares a number of catalytic properties with Fpg. In addition, endo VIII specifically bound to oligodeoxynucleotides containing a reduced AP site with a stoichiometry of 1:1 for protein to DNA with an apparent equilibrium dissociation constant of 3.9 nM. Like Fpg and endo III, the DNase I footprint was small with contact sites primarily on the damage-containing strand; unlike Fpg and endo III, the DNA binding of endo VIII to DNA was asymmetric, 3' to the reduced AP site.