An amidase acting on (R,S)-piperazine-2-tert-butylcarbox-amide was purified fromPseudomonas azotoformansIAM 1603 and characterized. The enzyme actedS-stereoselec-tively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (S)-piperazine-2-carboxylic acid. N-terminal and internal amino acid sequences of the enzyme were determined. ThegeneencodingtheS-stereoselective piperazine-2-tert-butylcarboxamide amidase was cloned from the chromo-somal DNAof the strain and sequenced.