The proximal region of the high-molecular-weight glutenin promoter of the Dx5 gene (PrHMWG-Dx5) carries an atypical bifactorial endosperm box containing two cis-acting elements, namely a G-box like motif followed by a prolamin-box motif (Pb1). Transient expression assays in maize endosperm indicate that a promoter fragment containing at least the G-box like element is necessary and sufficient for maximal expression of the HMWG-Dx5 promoter. In transformed maize, we have shown that a 89 bp sequence bearing the bifactorial endosperm box behaves like a functional cis-acting unit. Its repetition in tandem confers a strong specific additive effect specifically in endosperm tissue. In contrast, the fusion of the activation sequences 1 (as-1) and 2 (as-2) of the cauliflower mosaic virus (CaMV) 35S promoter with HMWG-Dx5 derived promoter sequences deregulates its activity in transformed maize. By gel mobility shift assays we have demonstrated that the G-box like motif may alternatively bind two protein groups which have the same DNA-binding affinities as the transcription factors of either the Opaque2 (O2) family and/or the ASF-1 family.