Different mechanisms have been proposed previously for the biodegradation of monomethyl sulphate (MMS) in Agrobacterium sp. and Hyphomicrobium sp. Sulphate liberation from MMS in Agrobacterium sp. M3C was previously shown to be O2-dependent, whereas in several Hyphomicrobium spp. the initiating step has been considered hitherto to be hydrolytic and catalysed by methyl sulphatase. In the present study, Agrobacterium and Hyphomicrobium strains were compared for their ability to oxidize MMS and its potential metabolites in the oxygen electrode. MMS-grown Agrobacterium sp. M3C and Hyphomicrobium sp. MS223 oxidized MMS with consumption of 0.5 mol O2 per mol of substrate, but they were unable to oxidize methanol. By repeatedly challenging MMS-grown Hypomicrobium with MMS in the electrode chamber, all the O2 in the electrode became exhausted, at which point SO4(2-) liberation stopped although excess MMS was available. SO4(2-) release resumed immediately when O2 was re-admitted to the electrode chamber. Thus liberation of SO4(2-) from MMS in the oxygen electrode was dependent on the continuing availability of O2. Hyphomicrobium sp. MS223 therefore closely resembled Agrobacterium sp. M3C in its obligatory requirement for O2 in MMS degradation. Unlike Agrobacterium sp. M3C, Hyphomicrobium sp. MS223 was able to grow on methanol and methanol-grown cells oxidized methanol (0.5 mol O2 per mol of substrate) but not MMS. Cyclopropanol, an inhibitor of methanol dehydrogenase, abolished oxidation of methanol by methanol-grown Hyphomicrobium sp. MS223 but did not affect oxidation of MMS by MMS-grown cells. Thus Hyphomicrobium sp. MS223 expresses enzymes for oxidation of methanol when needed for growth on this compound, but not when grown on MMS.(ABSTRACT TRUNCATED AT 250 WORDS)