HvGAMYB, a MYB transcription factor previously shown to be expressed in barley aleurone cells in response to gibberellin during germination, also has an important role in gene regulation during endosperm development. The mRNA was detected early (10 DAF) in the seeds where it accumulates, not only in the aleurone layer, starchy endosperm, nucellar projection and vascular tissue, but also in the immature embryo as shown by in situ hybridization analysis. The HvGAMYB protein, expressed in bacteria, binds to oligonucleotides containing the 5'-TAACAAC-3' or 5'-CAACTAAC-3' sequences, derived from the promoter regions of the endosperm-specific genes Hor2 and Itr1, encoding a B-hordein and trypsin-inhibitor BTI-CMe, respectively. Binding is prevented when these motifs are mutated to 5'-TgACAAg-3' and 5'-CgACTgAC-3'. Transient expression experiments in co-bombarded developing endosperms demonstrate that HvGAMYB trans-activates transcription from native Hor2 and Itr1 promoters through binding to the intact motifs described above. Trans-activation of the Hor2 promoter also requires an intact prolamine box (PB). This suggests that HvGAMYB interacts in developing barley endosperms with the PB-binding factor BPBF, an endosperm-specific DOF transcriptional activator of the Hor2 gene. The in vivo interaction experiment between HvGAMYB and BPBF was done in the yeast two-hybrid system, where HvGAMYB potentiates the BPBF trans-activation capacity through interaction with its C-terminal domain.