N-Nitrosamine formation by various intestinal bacteria was investigated. N-Nitroso-dimethylamine (NDMA) formation by viable resting cells of Escherichia coli A10 was proportional to the incubation time and the enzyme concentration, while boiled cells were incapable of nitrosation. The enzyme was optimal at pH 7.5 and showed about the same specificities for dimethylamine, diethylamine, dibutylamine, di-isobutylamine, piperidine and pyrrolidine, but high specificity for morpholine. The intestinal bacteria harbouring nitrosating enzyme were mainly aerobic, i.e., Escherichia coli, Proteus morganii, Klebsiella pneumonia and Pseudomonas aeruginosa. Only one of the 32 anaerobic intestinal bacterial species, i.e., Peptococcus asaccharolyticus, was positive. The enzyme activities of these nitrosating bacteria covered a range of 0.06-0.90 nmol NDMA formed per hour per mg protein. These results support the theory of enzymatic catalysis of N-nitrosamine formation by microorganisms and suggest the possibility of endogenous nitrosation in the digestive tract.