Because of its characteristics, the comet assay has been used to evaluate the ability of virtually any type of eukaryotic cell to repair different kinds of DNA damage, including double and single strand breaks and base damage. The ability to detect excision repair sites using the alkaline version can be enhanced by the inclusion of repair inhibitors, DNA synthesis inhibitors, or chain terminators. In this sense, we evaluated the ability of hydroxyurea (HU) and cytosine arabinoside (Ara-C), for detecting lesions produced by the alkylating agents ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) in three different cell systems. Two hundred cells for experimental point were analyzed in the alkaline version of the comet assay, and the results are evidences of the utility of the assay to detect alkylation of bases in the cells lines MRC-5 and TK-6, as the treatment with HU +Ara-C significantly increases both the basal and induced frequency of DNA damage. The use of whole blood, although it detected the effects of MMS, with and without repair inhibitors, failed to detect the effect of the selected dose of EMS and does not permit detection increases in the background level. © 2007 Springer Science+Business Media B.V.