The maize opaque 2 (o2) mutation is known to have numerous pleiotropic effects. Some polypeptides have their expression depressed while others are enhanced. The best characterized effects of the o2 mutation are those exerted on endosperm genes encoding the storage protein class of the 22 kDa alpha-zeins and the ribosome inactivating protein b-32. The Opaque 2 (O2) locus encodes a basic domain-leucine zipper DNA-binding factor, O2, which transcriptionally regulates these genes. In the maize-related grass Coix lacryma-jobi, an O2-homologous protein regulates the 25 kDa alpha-coixin family. We show in this paper that O2 transcriptionally regulates the structurally and developmentally different class of the beta-prolamins. A new O2-binding box was identified in beta-prolamin genes from maize and Coix that, together with the boxes previously identified in other endosperm expressed genes, forms a curious collection of O2 cis elements. This may have regulatory implications on the role of O2 in the mechanism that controls coordinated gene expression in the developing endosperm. Considering that the O2 locus controls at least three distinct classes of genes in maize endosperm, we propose that the O2 protein may play a more general role in maize endosperm development than previously conceived.