N-carbamoyl-L-cysteine amidohydrolase (NCC amidohydrolase) was purified and characterized from the crude extract of Escherichia coli in which the gene for NCC amidohydrolase of Pseudomonas sp. strain ON-4a was expressed. The enzyme was purified 58-fold to homogeneity with a yield of 16.1% by three steps of column chromatography. The results of gel filtration on Sephacryl S-300 and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 45-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 9.0 and 50 degreesC, respectively. The enzyme required Mn2+ ion for activity expression and was inhibited by EDTA, Hg2+ and sulfhydryl reagents. The enzyme was strictly specific for the L-form of N-carbamoyl-amino acids as substrates and exhibited high activity in the hydrolysis of N-carbamoyl-L-cysteine as substrate. These results suggested that the NCC amidohydrolase is a novel L-carbamoylase, different from the known L-carbamoylases.