The 3' --> 5'-exonucleolytic activity of human apurinic/apyrimidinic endonuclease I (APEI) on mispaired DNA at the 3'-termini of recessed, nicked or gapped DNA molecules was analyzed and compared with the primary endonucleolytic activity. We found that under reaction conditions optimal for AP endonuclease activity the 3' --> 5'-exonuclease activity of APE I manifest, only at enzyme concentration elevated by 6-7 orders of magnitude. This activity does not show a preference to mismatched compared to matched DNA structures as well as to nicked or gapped DNA substrates in comparison to recessed ones. Therefore. the 3' --> 5'-exonuclease activity associated with APE1 can hardly be considered as key mechanism that improves fidelity of DNA repair. (C) 2002 Elsevier Science (USA). All rights reserved.