Evidence is presented for the presence in propanesulfonate-grown Comamonas acidovorans strain P53 of a cytoplasmically located sulfonatase that does not sediment at 100,000 x g. This enzyme catalysed the sulfonate-dependent oxidation of NADH or NADPH, indicating a monooxygenase that effects the addition of molecular oxygen to C(3)-C(6) 1-alkanesulfonates. Enzyme activity was proportional to protein concentration only above approximately 2 mg cytoplasmic fraction protein ml(-1), suggesting that the sulfonatase is a multicomponent enzyme, possibly comparable with methanesulfonate monooxygenase. Enzyme activity was strongly inhibited by divalent metal-chelating agents, but was insensitive to cyanide and azide. Sulfite released from sulfonates by Comamonas acidovorans was oxidized by an unusual sulfite dehydrogenase. This was purified approximately 230-fold and was shown to have a molecular mass of 74.4 kDa, comprising two or more subunits. The enzyme activity was specific in vitro for ferricyanide as an electron acceptor and, unlike other bacterial sulfite dehydrogenases, did not contain native cytochrome c or reduce added cytochrome c. It was a basic protein, insensitive to chloride and sulfate, and exhibited a K(m) for sulfite of approximately 45 &mgr;M.