L-Cysteine which is widely used in food additives, nutritional infusions, and cosmetics and medicines has mainly been produced from hydrolysates of hair by acid or alkali. As an alternative to this traditional method, a new microbial conversion method for L-cysteine production from a chemically synthesized precursor, D,L-2-amino-∆²-thiazoline-4-carboxylic acid (D,L-ATC), using Pseudomonas species was developed. From the studies on the microbial conversion process of D,L-ATC to L-cysteine in several Pseudomonas strains by several groups, it was found that there are two pathways via S-carbamoyl-L-cysteine (L-SCC, pathway 1) and via N-carbamoyl-L-cysteine (L-NCC, pathway 2) in the microbial conversion process. We isolated and identified the genes for ATC hydrolase and NCC amidohydrolase, which are involved in pathway 2 in Pseudomonas sp. ON-4a. The ATC hydrolase and NCC amidohydrolase expressed in Escherichia coli were purified and characterized. In this study, we clarified the molecular basis of the microbial conversion of D,L-ATC to L-cysteine. We propose that L-cysteine production from D,L-ATC can be effectively carried out by two continuous reactions using recombinant ATC hydrolase and NCC amidohydrolase.