The enantioselective amidase from Rhodococcus sp. strain R312 was produced in Escherichia coli and was purified in one chromatographic step. This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides. The optimum working pH values were 7 with neutral amides and 8 with alpha-aminoamides. The reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the Km(amide) values (e.g., Km(amide) = 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide). Moreover, very high turnover numbers (kcat) were obtained with linear aliphatic amides (e.g., kcat = 333 s-1 with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered. Carboxylic acids, alpha-amino acids, and methyl esters were not acyl donors or were very bad acyl donors. Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors. On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g., KmNH2OH = 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively). No acyl acceptors except water and hydroxylamine were found. Finally, the purified amidase was shown to be L-enantioselective towards alpha-hydroxy- and alpha-aminoamides.