Osmotin-like protein (OLP) is a neutral isoform in the group 5 pathogenesis-related (PR) tobacco proteins. The OLP gene, like the basic PR protein genes, is constitutively expressed in tobacco roots and cultured cells. OLP is not naturally present in intact healthy leaves, but ethylene treatment induces a high accumulation there. To study the mechanism of OLP gene expression as induced by ethylene, we cloned the gene from Nicotiana sylvestris, an ancestor of N. tabacum. Sequence analysis showed that it has no intron and that its promoter region contains two AGCCGCC sequences that are conserved in most basic PR-protein genes. The function of the AGCCGCC sequences in transgenic tobacco plants that harbor the wild and mutated OLP promoter::GUS fusion genes was analyzed. Mutation in the AGCCGCC sequences clearly inhibited the GUS expression induced by ethylene, indicative that the AGCCGCC sequence(s) is a DNA element(s) responsive to ethylene. An EREBP2 protein, isolated as one of the proteins binding to the AGCCGCC sequence of the tobacco beta-1,3-glucanase gene, also was found to bind to the AGCCGCC sequence(s) of OLP gene. These results suggest that the ethylene-induced expression of OLP is regulated by a trans-acting factor(s) common to basic PR-proteins.