Ionizing radiation is one of the most potent inducers of O6-methylguanine-DNA methyltransferase (MT) in rat liver in vivo. In this study we show that MT is readily induced in C3H/10T1/2 cells in culture, which provides a system more amenable to determining the molecular events involved in the induction of this repair enzyme. Maximal induction (2- to 3-fold) was observed in logarithmically growing cells 48 h after a dose of 200 rad, similar to the optimal induction time seen in rat liver in vivo. The absolute level of MT observed in C3H/10T1/2 cells which had been at confluence for 24 h was less than in cells in log growth but was still inducible by X-rays, exhibiting an approximately 2-fold increase over unirradiated cells similar to MT induction in logarithmically growing cells. Irradiating cells under anaerobic conditions (saturated N2) abolished MT induction by 100 rad. Cells irradiated with 200 rad under anaerobic conditions exhibited approximately 70% inhibition of induction compared with aerobically irradiated cells. The possibility that MT may be partially inactivated by interaction with radicals produced by ionizing radiation is discussed.