We show that a 61 bp fragment derived from the promoter region of the tobacco class I beta-1,3-glucanase GLB gene enhances transcription in Nicotiana plumbaginifolia protoplasts independent of orientation relative to the start of transcription. This fragment leads to a cooperative stimulation of transcription when combined with the cauliflower mosaic virus 35S as-1 enhancer element. The GLB enhancer contains two copies of the sequence AGCCGCC, which is conserved in several genes showing expression patterns similar to the GLB gene, as well as a sequence identical at 6 of 7 bp. Point mutations in these three sequences eliminate the enhancer activity of the 61 bp fragment. Nuclear extracts prepared from leaves of tobacco plants contain one or more putative transcription factors that interact specifically with the GLB enhancer. This factor was much less abundant in nuclear extracts prepared from upper leaves of untreated tobacco plants than in nuclear extracts prepared from upper leaves of ethylene-treated plants or from lower leaves. Since beta-1,3-glucanase genes are expressed at very low levels in upper leaves of tobacco plants, at higher levels in lower leaves, and are induced in all leaves after treatment of plants with the stress hormone ethylene, we conclude that the enhancer element interacts with one or more transcription factors whose binding activity is correlated with gene expression in vivo.