A carbamoylase enzyme was purified from a cell-free extract of Agrobacterium sp. with an overall yield of 81%. It was judged to be homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight of 38,000 daltons. Further studies on the native enzyme suggested that the active enzyme was present as a dimer, with a pl of 5.5. It was able to cleave a variety of N-carbamoyl substrates, but was strictly D(-) specific. It was found to have a K-m of 0.82 mM and a V-max of 31 U mg(-1) for D(-) N-carbamoyl hydroxyphenylglycine in the presence of 10 mM dithiothreitol. It showed no metal ion requirements but was inhibited by iodoacetic acid and iodoacetamide, both thiol reagents. The N-terminal amino acid sequence of the enzyme was elucidated.