A recombinant Escherichia coli containing a gene from Pseudomonas putida NTU-8 for producing creatinase and a signal peptide from Aeromonas hydrophila for transporting the enzyme into the periplasmic space was incubated in LB medium by employing IPTG as an inducer. The cell growth and creatinase production were optimized at the induction time of 1.75 h by adding 100 mg/L of IPTG in a batch fermentor. By employing a series of separation steps such as centrifugation, cell breakage, ultrafiltration and FPLC purification, 76.3% of enzyme recovery with 2-fold enhancement of the specific creatinase activity from the broth was obtained. By using SDS-PAGE and IEF electrophoresis, the molecular weight of 48.8 kDa and pI of 4.52 for the enzyme was estimated. The maximum activity for the crude and purified creatinases was at 37degreesC and pH around 7.7. Good enzyme thermal stability below 37degreesC at pH 7.7 or at the pH between 5.5 and 9.4 at 37degreesC was found. Inhibition of both enzymes by Ni2+, Cu2+, Ag+ and Hg2+ ions and good substrate specificity toward creatine were also reported.