The expression of tobacco class I chitinase genes is effectively induced by a fungal elicitor in suspension-cultured cells. A putative cis-acting elicitor-responsive element (EIRE) was identified previously in the promoter of the class I chitinase gene, CHN5O. To confirm that the EIRE sequence directly mediates the regulation of gene expression by the elicitor, I constructed a deleted promoter that controlled a reporter gene for beta-glucuronidase (gus) and examined expression of the construct in transgenic tobacco calli. Both expression and responsiveness to the elicitor disappeared, when the region of the promoter that included the EIRE sequence had been deleted. To define the specific sequence within the EIRE that interacts with nuclear factor(s), a gel mobility shift assay was performed with wild-type and mutated elements. Results of binding and competition experiments revealed that the nuclear factor(s) bound specifically to the sequence motif, (-534)GGTCANNNAGTC(-523), and that both of the repeated sites were involved in the binding of the nuclear factors. Moreover, the binding was influenced by the distance between the two repeated sites. In addition, the elicitor-inducible activity of the binding to this motif was reduced in nuclear extracts prepared from the cells that had been treated with cycloheximide or staurosporine.