Nitrilase from Rhodococcus ATCC 39484 was found to consist of two species of Mr 40,258 +/- 2 and 40,388 +/- 2 Da. When the enzyme was incubated with nitrile substrates and the reaction quenched with acid, higher Mr species were observed. The mass differences were consistent with addition of a substrate molecule to each species. These results represent the first reported demonstration that this, or any other nitrilase forms a covalent intermediate with its substrates. The observation that the intermediate, suggested to be either a thioimidate or an acylenzyme, can be trapped by acidification indicates that the rate of breakdown of the intermediate is rate-limiting.