Ethylene-inducing xylanase (EIX) elicits plant defense responses in certain tobacco (Nicotiana tabacum) and tomato cultivars in addition to its xylan degradation activity. It is not clear, however, whether elicitation occurs by cell wall fragments released by the enzymatic activity or by the xylanase protein interacting directly with the plant cells. We cloned the gene encoding EIX protein and overexpressed it in insect cells. To determine the relationship between the two activities, substitution of amino acids in the xylanase active site was performed. Substitution at glutamic acid-86 or -177 with glutamine (Gln), aspartic acid (Asp), or glycine (Gly) inhibited the beta-1-4-endoxylanase activity. Mutants having Asp-86 or Gln-177 also lost the ability to induce the hypersensitive response and ethylene biosynthesis. However, mutants having Gln-86, Gly-86, Asp-177, or Gly-177 retained ability to induce ethylene biosynthesis and the hypersensitive response. Our data show that the xylanase activity of EIX elicitor can be separated from the elicitation process, as some of the mutants lack the former but retain the latter.