Glyoxal (ethanedial) is an increasingly used industrial chemical that has been found to be mutagenic in bacteria and mammalian cells. In this study, the reactions of glyoxal with 2‘-deoxyguanosine, 2‘-deoxyadenosine, 2‘-deoxycytidine, cytidine, thymidine, and calf thymus DNA have been studied in aqueous buffered solutions. The nucleoside adducts were isolated by reversed-phase liquid chromatography and characterized by their UV absorbance and 1H and 13C NMR spectroscopic and mass spectrometric features. The reaction with 2‘-deoxyguanosine gave one adduct, the previously known 3-(2‘-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one adduct. The reaction of 2‘-deoxyadenosine with glyoxal resulted in the formation of a previously not reported N6-(hydroxyacetyl)-2‘-deoxyadenosine adduct. In the reaction of glyoxal with 2‘-deoxycytidine and cytidine at neutral conditions and 37 °C, 5-hydroxyacetyl pyrimidine derivatives were obtained. When the cytidine reaction was performed at pH 4.5 and 50 °C, the 5-hydroxyacetyl derivative of uridine was formed through deamination of cytidine−glyoxal. Adducts in the thymidine reaction could not be detected. In the reaction of glyoxal with calf thymus DNA, the 2‘-deoxyguanosine−glyoxal and 2‘-deoxyadenosine−glyoxal adducts were obtained, the former being the major adduct.