Cellulomonas flavigena NTOU 1 could grow well and show higher chitinase activity in the modified salts-pectin broth additionally containing 1% (w/v) shrimp shell powder and 0.5% yeast extract. When crude enzyme solution was concentrated and purified through ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration on fast protein liquid chromatography, a chitinase was isolated. The enzyme displayed a specific activity of 232.9 U mg(-1) protein and gave a single band on SDS-PAGE. The molecular weight of the purified chitinase was estimated at around 34.2 kDa by gelfiltration and 32.5 kDa by SDS-PAGE. The optimum pH and temperature were 10.0 and 50 degrees C, respectively. The protein was stable in the pH range from 6-10 and up to 45 degrees C. Fe3+, Hg2+, N-ethylmaleimide, monoiodoacetate, and beta-mercaptoethanol inhibited enzyme activity while Na+, Zn2+, and Mg2+ caused activation. The chitinase showed a K-m value of 0.15 mM against 4-methylumbelliferyldiacetylchitobiose. The chitinase degraded substrates in an exo-splitting manner as an exo-N,N’-diacetylchitobiohydrolase.