Recombinant class V chitinases from Nicotiana tabacum and Arabidopsis thaliana (NtChiV and AtChiC) were produced by the Escherichia coli expression system, and the antifungal activity of the enzymes was investigated using the hyphal extension inhibition assay on agar plates with Trichoderma viride as the test fungus. The activity of NtChiV was found to be much higher than that of AtChiC. The inactive mutants of both enzymes, in which the individual catalytic acids were mutated to glutamine, were also tested by the same assay system. The activity was impaired by the mutation, indicating that the hydrolytic activity contributes to the antifungal action of the enzymes. However, the activity of the enzymes toward glycol chitin substrate was not proportional to the antifungal activity, indicating that the hydrolytic activity does not exclusively contribute to the antifungal action. X-ray crystal structures of these enzymes revealed that the aglycon-binding region of NtChiV consists of a number of polar side chains but not in AtChiC. Polarity of the surface of substrate-binding cleft could be another factor controlling the antifungal action of class V chitinases.