N -Nitrosodialkylamines, activated metabolically by cytochrome P450, possess mutagenic and carcinogenic activity. In this study, the hydroxyl radical, generated from Fenton's reagent, was used as an oxidant for the activation of the N -nitrosodialkylamines. Ethyl acetate extract from the reaction mixture which included Fe²⁺-Cu²⁺-H₂O₂ and N -nitrosodialkylamines; N -nitrosodimethylamine (NDM), N -nitrosodiethylamine (NDE), N -nitrosodipropylamine (NDP), N -nitrosodibutylamine (NDB), N -nitroso- N -methylpropylamine (NMP), N -nitroso- N -methylbutylamine (NMB), were assayed for their mutagenicity in Salmonella typhimurium ( S. typhimurium ) TA1535 and Escherichia coli ( E. coli ) WP2 uvr A. Although Fenton's reagent (Fe²⁺-H₂O₂) alone did not activate NMB, the addition of the copper ion to the reaction with Fenton's reagent (Fe²⁺-Cu²⁺-H₂O₂) resulted in the production of mutagens. While the extracts of the reaction of NDM or NDE with Fe²⁺-Cu²⁺-H₂O₂ were not mutagenic, those of NMP, NDP, NMB, or NDB with Fe²⁺-Cu²⁺-H₂O₂ were mutagenic in both S. typhimurium TA1535 and E. coli WP2 uvr A. These results demonstrate that a direct-acting mutagen was formed from N -nitrosodialkylamines, with alkyl chains longer than propyl, by the oxidation in the Fe²⁺-Cu²⁺-H₂O₂ system.