We have assayed in extracts of HeLa cells the amount of acceptor protein that removes O6-methylguanine adducts from alkylated DNA. Cells were treated with single or multiple nontoxic doses of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and the extracts were analyzed up to 32 h after the last exposure. The acceptor activity assayed immediately (1 h) after single exposures decreases linearly with dose indicating that the acceptor protein is used up by endogenous O6-methylguanine adducts in a stoichiometric reaction. Multiple exposures, assayed 8-24 h after the last exposure, increase the amount of acceptor protein in a dose dependent fashion followed by a decrease above a cumulative dose of 100 ng/ml. Under conditions of maximum induction, there are about 300,000 acceptor protein sites per cell, approximately 3 fold above the constitutive level. Both in adapted and unadapted cells the methyl group from O6-methylguanine adducts in the alkylated DNA is transferred to cysteine residues of the acceptor protein(s).