The promoter of the wheat low-molecular-weight glutenin (LMWG1D1) gene contains a cis element called the GCN4 like motif (GLM) which has low homology to one class of binding site for the maize endosperm-specific b-ZIP transcription factor Opaque-2 (O2). Previous work has shown that the GLM element interacts with the nuclear factor ESBFII during wheat endosperm development at the time of maximum transcription of the LMWG1D1 gene. In this paper we demonstrate that O2 binds to the GLM element and can activate high levels of transcription from the wheat GLM in transient assays in plant protoplasts and in yeast cells. Lower levels of O2 activation through the GLM element in yeast containing a defective GCN4 gene showed that GCN4 was necessary for high levels of O2 transcriptional activation, indicating that O2 may need to heterodimerise with GCN4 to activate transcription in yeast. These observations provide evidence that the GLM represents a new type of O2 DNA-binding site, and support a postulate that an O2 homologue may activate endosperm-specific expression of wheat storage protein genes.