N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced repair (unscheduled) DNA synthesis was studied in intact HeLa cells as well as in the cell lysate. The incorporation of dTTP[methyl-3H] in vitro by the cell lysate was dependent on the dose of MNNG, and as linear for 2 min, reaching the maximum at 4 min. The optimum conditions were the same as for UV-induced in vitro repair synthesis. The presence of hydroxyurea and arabinofuranosyl cytosine during in vivo incubation with MNNG was necessary for enhancement of in vitro DNA synthesis. This MNNG-induced in vitro DNA synthesis was inhibited by aphidicolin, but not by 2',3'-dideoxythymidine-5'-triphosphate. It thus appears that DNA polymerase alpha participates in MNNG-induced unscheduled DNA synthesis in vitro.