The purpose of this investigation was to obtain a comparative measure of DNA repair synthesis in hormone-dependent normal mammary tissue and hormone-independent D1 hyperplastic alveolar nodule (HAN) outgrowth. Treatment of mammary fragments in culture with 5 mM hydroxyurea inhibits 98% of the semiconservative DNA synthesis. Treatment of mammary fragments with methyl methanesulfonate in the presence of hydroxyurea results in a 4- to 7-fold higher incorporation of [3H]deoxythymidine into the mammary cell DNA than does treatment with hydroxyurea alone. This hydroxyurea-resistant alkylating agent-induced [3H]deoxythymidine incorporation was studied by CsCl density gradient centrifugation and has been found to represent DNA repair replication. Similar levels of repair replication were found in both normal and preneoplastic D1-HAN outgrowth. Autoradiographic analysis of mammary fragments and D1-HAN outgrowth treated with methyl methanesulfonate plus hydroxyurea revealed that 30 to 50% of the epithelial cell nuclei were lightly labeled. No detectable repair synthesis was found in fat or stromal cells. The average number of grains per labeled nuclei was the same for both explants. These results suggest that a reduced DNA repair capacity is not associated with the increased sensitivity of D1-HAN outgrowth to the tumorigenic effect of chemical carcinogens.