N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of 18 O 2 and H 2 18 O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O 2 . Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH 3 OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial ␣-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.N-Nitrosodimethylamine (NDMA) is present in groundwater and drinking water primarily as a by-product of wastewater and drinking water disinfection and from past military testing and disposal of 1,1-dimethylhydrazine, a component of liquid rocket propellant that contained NDMA as an impurity (9,20,21). NDMA is a potent mutagen and a suspected human carcinogen, so its presence in drinking water is of significant concern (1, 22, 34, 36). Although there is no federal drinking water standard for NDMA, the United States Environmental Protection Agency has estimated that concentrations exceeding 0.7 ng/liter may significantly increase cancer risk (34), and the California Office of Environmental Health Hazard Assessment recently set a draft public health goal of 3 ng/liter (24).The metabolism of NDMA in animals and its toxicological effects have been widely studied (1,24,36). The mammalian metabolism of NDMA is initiated by the cytochrome P450-dependent mixed-function oxidase system and follows either a demethylation (␣-hydroxylation) or a denitrosation pathway depending on the site of oxidative attack (1, 32, 36) (Fig. 1). The demethylation route (Fig. 1A) results in the formation of the methyldiazonium ion, a strong alkylating agent which is thought to account for much of the carcinogenic activity of NDMA. This ion either binds to macromolecules or spontaneously disassociates to methanol and nitrogen gas (29, 36). The denitrosation pathway (Fig. 1B) results in the formation of nitrite, methylamine, and formaldehyde as metabolites (36).In contrast to the extensive data from animal studies, there is relatively l...