Pseudomonas sp. strain NS671, which produces L-amino acids asymmetrically from the corresponding racemic 5-substituted hydantoins, harbored a plasmid of 172 kb. Curing experiments suggest that this plasmid, designated pHN671, is responsible for the conversion of 5-substituted hydantoins to their corresponding L-amino acids by strain NS671. DNA fragments containing the genes involved in this conversion were cloned from pHN671 in Escherichia coli by using pUC18 as a cloning vector. The smallest recombinant plasmid, designated pHPB12, contained a 7.5-kb insert DNA. The nucleotide sequence of the insert DNA was determined, and three closely spaced open reading frames predicted to encode peptides with molecular masses of 75.6, 64.9, and 45.7 kDa were found. These open reading frames were designated hyuA, hyuB, and hyuC, respectively. Cell extracts from E. coli carrying deletion derivatives of pHPB12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gene products of hyuA, hyuB, and hyuC were identified. The functions of these gene products were also examined with the deletion derivatives. The results indicate that both hyuA and hyuB are involved in the conversions of D- and L-5-substituted hydantoins to corresponding N-carbamyl-D- and N-carbamyl-L-amino acids, respectively, and that hyuC is involved in the conversion of N-carbamyl-L-amino acids to L-amino acids.