To characterize a novel dimethoate amidohydrolase from Sphingomonas sp. DC-6. A gene, dmhA, encoding the dimethoate amidohydrolase responsible for transforming dimethoate to dimethoate carboxylic acid and methylamine, was cloned from Sphingomonas sp. DC-6. Sequence analysis and molecular modeling indicate that DmhA shares 31-57 % amino acid sequence identities with other functionally confirmed amidohydrolase. DmhA was expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. The purified DmhA could hydrolyze 4-acetaminophenol, dimethoate and propanil. DmhA activity was optimal at 30 A degrees C and pH 7.5. Hg2+, Zn2+, Cu2+, Cd2+, Tween 80, Triton X-100 or SDS strongly inhibited its activity. The K (m) and k (cat) values of DmhA for dimethoate are 0.02 mM and 1.2 s(-1), respectively. DmhA was confirmed to be a novel dimethoate amidohydrolase which could eliminate the toxicity of dimethoate, providing a novel gene resource for the development of pesticide-degrading enzyme preparation and mechanistic study of dimethoate hydrolysis.