An isolate of a Pseudomonas sp. uses the l-NCC (N-carbamoyl-l-cysteine) pathway to convert dl-2-amino-Delta(2)-thiazoline-4-carboxylic acid (dl-ATC) to l-cysteine. Genes encoding ATC racemase (AtcA), l-ATC hydrolase (AtcB) and l-NCC amidohydrolase (AtcC), involved in this pathway, were cloned from the Pseudomonas sp. and expressed in Escherichia coli BL21 via pET-28a(+). The resulting enzymes were purified, their functions identified, and their biochemical properties are described. In vitro catalysis experiments, using these enzymes, revealed that the bioconversion rate of l-cysteine from dl-ATC in the presence of AtcA was more efficient than in the absence of AtcA. This is the first report describing simultaneous cloning and expression of atcA, atcB and atcC and characterization of their enzymes for l-cysteine production from dl-ATC via the l-NCC pathway, enabling the complete l-NCC pathway to be elucidated.