Isonitrile containing an N triple bond C triple bond was degraded by microorganism sp. N19-2, which was isolated from soil through a 2-month acclimatization culture in the presence of this compound. The isonitrile-degrading microorganism was identified as Pseudomonas putida. The microbial degradation was found to proceed through an enzymatic reaction, the isonitrile being hydrated to the corresponding N-substituted formamide. The enzyme, named isonitrile hydratase, was purified and characterized. The native enzyme had a molecular mass of about 59 kDa and consisted of two identical subunits. The enzyme stoichiometrically catalyzed the hydration of cyclohexyl isocyanide (an isonitrile) to N-cyclohexylformamide, but no formation of other compounds was detected. The apparent K(m) value for cyclohexyl isocyanide was 16.2 mm. Although the enzyme acted on various isonitriles, no nitriles or amides were accepted as substrates.