The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene from Agrobacterium radiobacter NRRL B11291 has been successfully cloned and expressed in Escherichia coli. Subcloning of the D-carbamoylase gene into different types of vectors and backgrounds of E. coli strains showed that the optimal expression level of D-carbamoylase was achieved in a ColE1-derived plasmid with a 150-fold increase in specific enzyme activity compared to that in a pSC101-derived plasmid. In addition, the recombinant plasmids were very stable in the E. coli strain ATCC11303 but not in JCL1258 tested here. Employing the recombinant E, coli strain DH5 alpha/pAH61 for D-p-hydroxyphenylglycine production showed that the cell was capable of transforming N-carbamoyl-D-hydroxylphenylglycine to D-p-hydroxyphenylglycine with a molar conversion yield of 100% and a production rate of 1.9 g/(L h). In comparison with A. radiobacter NRRL B11291, this productivity approximates a 55-fold increase in D-hydroxyphenylglycine production. This result suggests the potential application of recombinant E. coli strains for the transformation reaction.