We used an anti-trimethylamine-N-oxide reductase (EC 1.6.6.9) serum and different immunological techniques (Ouchterlony, rocket immunoelectrophoresis, immunoblotting) to show that dimethylsulphoxide (DMSO), tetrahydrothiophene 1-oxide (THTO) and pyridine N-oxide (PNO) were effective inducers of the inducible form of trimethylamine N-oxide reductase. We confirmed this genetically and biochemically using a strain in which phage MudII 1734 carrying lacZ was inserted into torA, the structural gene for inducible trimethylamine-N-oxide reductase. By subcellular fractionation and quantitation with rocket immunoelectrophoresis, we showed that the enzyme was principally localized in the periplasmic fraction. Constitutive trimethylamine-N-oxide reductase was localized in the membrane fraction and, like the inducible enzyme showed a broad specificity with respect to various compounds such as DMSO, THTO and PNO. Apart from their immunological properties, the two enzymes could be clearly differentiated by their temperature stability.