THE repair of DNA requires the removal of abasic sites, which are constantly generated in vivo both spontaneously(1) and by enzymatic removal of uracil(2), and of bases damaged by active oxygen species, alkylating agents and ionizing radiation The major apurinic/apyrimidinic (AP) DNA-repair endonuclease in Escherichia coli is the multifunctional enzyme exonuclease In, which also exhibits 3’-repair diesterase, 3’-->5’ exonuclease, 3’-phosphomonoesterase and ribonuclease activities(5). We report here the 1.7 Angstrom resolution crystal structure of exonuclease III which reveals a 2-fold symmetric, four-layered ap fold,vith similarities to both deoxyribonuclease I-6 and RNase H-7. In the ternary complex determined at 2.6 Angstrom resolution, Mn2+ and dCMP bind to exonuclease III at one end of the alpha beta-sandwich, in a region dominated by positive electrostatic potential. Residues conserved among AP endonucleases from bacteria to man cluster within this active site and appear to participate in phosphate-bond cleavage at AP sites through a nucleophilic attack facilitated by a single bound metal ion.