In addition to the bZIP protein Opaque2 (O2), there are other maize endosperm nuclear proteins that recognize the O2 box in 22 kDa zein gene promoters. In an effort to understand the effect of these factors on 22 kDa zein expression, we have cloned one of these and identified it as the putative maize (Zea mays L.) orthologue of the wheat bZIP protein EmBP-1 (mEmBP-1). The mEmBP-1 protein exhibits 52% sequence identity and 68% similarity with the wheat protein and recognizes a similar spectrum of DNA sequences, albeit with slightly altered specificity. The mEmBP-1 gene exists as duplicate loci in maize on chromosomes 7S (mEmBP-1a) and 2L (mEmBP-1b). The mEmBP-1 genes are expressed in endosperm, embryo, immature ears, tassel, roots, and seedling shoots at low levels. Although mEmBP-1 binds to the O2 box from the 22 kDa zein gene promoter as a homodimer, it is unable to heterodimerize with O2. The mEmBP-1 protein can activate transcription from a truncated promoter containing a pentamer of the O2 site in yeast cells; however, it inhibited regulated transcription of a 22 kDa zein promoter in a transient expression assay using cultured maize endosperm cells.