The oxidation of cytosine in DNA by free radicals and other oxidants leads to an assortment of products including pyrimidine ring 5,6-saturated, 5,6-unsaturated, contraction, and fragmentation products. The formation of these products in cellular DNA may explain in part the preponderance of C to T transitions induced spontaneously and by H2O2 or ionizing radiation. Our studies have focused on the biological effects of two major 5,6-unsaturated oxidation products of cytosine: 5- hydroxycytosine and 5-hydroxyuracil. In the present work, we have attempted to study the repair of these two lesions by specifically incorporating them into cellular DNA upon incubation of cells with 5-hydroxy-2'deoxycytidine and 5-hydroxy-2'-deoxyuridine. Incubation of mouse L1210 cells with 250 M 5-hydroxy-2'-deoxycytidine led to the incorporation of this lesion to a level 20 times higher (43 lesions/10(5) cytosines) than base-line levels; however, there was no evidence for its repair following a 15-h chase. In contrast, we did not observe any significant incorporation of 5-hydroxy-2'-deoxyuridine into the DNA of L1210 cells but did observe an unidentified product, presumably an oxidation product. This unidentified pyrimidine was incorporated at a very high level (about 2000 lesions/10(5) cytosine residues) and then partially repaired in chase experiments.