The microbial degradation of nitriles is of interest for bioremediation and green chemistry. We demonstrated that the soil bacterium Rhodococcus sp. RHA1 utilizes a range of nitriles, including acetonitrile, as growth substrates. Proteomic analysis identified 13 proteins that were more abundant in acetonitrile-grown cells, including an aliphatic amidase and a protein with no known homologue. Purification of a nitrile hydratase (NHase) from acetonitrile-grown cells identified the unknown protein as the beta subunit of a two-subunit NHase. Sequence analysis revealed that the genes encoding the amidase (anhC) and the NHase (anhAB) occur in a 12.8 kbp cluster located on plasmid pRHL2. The anh gene cluster also encodes an acetyl-CoA hydrolase, transcriptional regulators, a putative cobalt transporter and a protein of unknown function. Striking features of the NHase include the amino acid sequence identity (32%) and large size (63 and 56 kDa) of the alpha and beta subunits, as well as the enzyme's metal ion content (one cobalt, two copper and one zinc). The enzyme possessed similar specificities for acetonitrile and propionitrile (k(cat)/K(m) approximately 7 mM(-1) s(-1)) followed by acrylonitrile and butyronitrile. We propose that this acetonitrile hydratase (ANHase) represents the first member of a previously unknown class of NHases.