Alkylating agents can produce a variety of biological effects in mammalian cells and organisms including toxicity, mutagenicity and malignant transformation. These agents react with oxygen and nitrogen atoms in DNA resulting in 12 products some of which are known to be eliminated from DNA by repair systems. One method of assessing the relative importance of a specific product in any of the biological effects of DNA alkylation would be to convert a cell line that is deficient in a particular repair function into a repair-proficient cell line and to determine whether this influences the magnitude of the effect. The cloning and expression in mammalian cells of the Escherichia coli DNA repair gene coding for the O6-alkylguanine-alkylphosphotriester dual alkyltransferase will be described. The E. coli gene product acts on damage produced in host cell DNA by treatment with methylnitrosourea, and reduces the toxicity and mutagenicity of this agent. The effects on the toxicity of a variety of other mono and bifunctional alkylating agents have also been assessed.