A method was developed for purification and crystallization of creatinase [creatine amidinohydrolase, EC 3.5.3.3] from Pseudomonas putida var. naraensis C-83. The purified preparation appeared homogeneous on disc electrophoresis and ultracentri-fugation and had a molecular weight of 94, 000. It was most active at pH 8 and stable between pH 6 and 8 for 24 hr at 37°. SDS-polyacrylamide gel electrophoresis indicated that the native enzyme was made up of two subunit monomers, the molecular weights of which were estimated to be 47, 000. Inhibition experiments suggested that a sulfhydryl group is located in or near the active site of the enzyme.