We have investigated the 3'-5'-exonuclease activity of phage T7 DNA polymerase for its usefulness as an approach for the detection of lesions in DNA. Unlike the T4 DNA polymerase-exonuclease, which is commonly used to map the position and frequency of lesions in very small DNA fragments, T7 DNA polymerase-exonuclease is able to hydrolyse almost completely the large fragments from KpnI-restricted mammalian DNA. However, we found that the exonuclease was also able to hydrolyse DNA containing several kinds of lesions: cyclobutane pyrimidine dimers, thymine glycols, and mono-adducts of 4'-hydroxymethyl-4,5',8-trimethylpsoralen and 5'-methyl-isopsoralen. Modifications of the reaction conditions did not significantly alter the extent of hydrolysis. These properties distinguish the T7 DNA polymerase-exonuclease from the T4 DNA polymerase-exonuclease and make the T7 DNA polymerase-exonuclease unsuitable for detecting several types of lesions in DNA.