An understanding of the contribution of reactive oxygen species to mutagenesis has been hampered by the vast number of different chemical modifications they cause in DNA. Even though many of these DNA alterations have been catalogued, the identification of specific lesions that cause mutations has depended on testing one modification at a time. In this study we present another approach to identify key mutagenic lesions from a pool of oxidatively modified nucleotides. dCTP was treated with an oxygen radical- generating system containing FeSO4, H2O2, and ascorbic acid. The modification products were separated by reverse-phase and anion-exchange HPLC and then incorporated by human immunodeficiency virus reverse transcriptase into a DNA that contains a target gene for scoring for mutations. One of the mutagenic species isolated was identified as 5-hydroxy-2'-deoxycytidine. It is incorporated efficiently into DNA and causes C → T transitions in Escherichia coli at a frequency of 2.5%, which is more mutagenic than any previously identified oxidative DNA lesion.