A reaction system was developed for the production of D-amino acids from D,L-5-monosubstituted hydantoins with a very slow rate of spontaneous racemization. For this purpose the D-hydantoinase and D-carbamoylase from Agrobacterium radiobacter NRRL B11291 were cloned in separate plasmids and expressed in Escherichia coli. The third enzyme, hydantoin racemase, was cloned from Agrobacterium tumefaciens C58. The hydantoin racemase amino acid sequence was significantly similar to those previously described. A reaction system consisting of recombinant Escherichia coli whole cell biocatalysts containing separately expressed D-hydantoinase, D-carbamoylase, and hydantoin recemase showed high substrate specificity and was effective toward both aliphatic and aromatic D,L-5-monosubstituted hydantoins. After optimizing reaction conditions (pH 8 and 50 degrees C), 100% conversion of D,L-5-(2-methylthioethyl)-hydantoin (15 mM) into D-methionine was obtained in 30 min.