Plant chitinases are pathogenesis-related proteins, which are believed to be involved in plant defense responses to pathogen infection. In this study, chitinase gene from barley was cloned and overexpressed in Escherichia coli. Chitinase (35 kDa) was isolated and purified. Since the protein was produced as insoluble inclusion bodies, the protein was solubilized and refolded. Purified chitinase exerted broad-spectrum antifungal activity against Botrytis cinerea (blight of tobacco), Pestalotia theae (leaf spot of tea), Bipolaris oryzae (brown spot of rice), Alternaria sp. (grain discoloration of rice), Curvularia lunata (leaf spot of clover) and Rhizoctonia solani (sheath blight of rice). Due to the potential of broad-spectrum antifungal activity barley chitinase gene can be used to enhance fungal-resistance in crop plants such as rice, tobacco, tea and clover. (c) 2006 Elsevier Inc. All rights reserved.