N-Carbamoyl-D-amino acid amidohydrolase was purified 119-fold, with 36% overall recovery from a cell-free extract of Comamonas sp. E222c. The purified enzyme was homogeneous as judged by SDS/PAGE. The relative molecular mass of the native enzyme was 120,000 and that of the subunit was 40,000. The purified enzyme hydrolyzed various N-carbamoyl-D-amino acids to D-amino acids, ammonia and carbon dioxide. N-Carbamoyl-D-amino acids having hydrophobic groups served as good substrates for the enzyme. The Km and Vmax values for N-carbamoyl-D-phenylalanine were 19.7 mM and 13.1 units/mg, respectively, and those for N-carbamoyl-D-p-hydroxyphenylglycine were 13.1 mM and 0.56 units/mg, respectively. The enzyme strictly recognized the configuration of the substrate and only the D-enantiomer of the N-carbamoyl amino acid was hydrolyzed. The enzyme activity was not significantly affected by N-carbamoyl-L-amino acids and ammonia. The enzyme was sensitive to thiol reagents and did not require metal ions for its activity. The enzyme did not hydrolyze N-carbamoyl-beta-alanine or N-carbamoyl-DL-aspartate suggesting that the enzyme is different from the N-carbamoylamide-hydrolyzing enzymes involved in the pyrimidine degradation pathway. The enzyme did not hydrolyze allantoin and allantoic acid, which are intermediates in purine degradation, N-carbamoylsarcosine and citrulline, suggesting that it is a novel N-carbamoylamide amidohydrolase.