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 18O2 and H(2)18O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O2. 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 (CH3OH) 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 alpha-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.