We established a high-throughput screening method for the DNA binding sequence of a eukaryotic transcription factor by using the bead display system with emulsion PCR and flow cytometry and applied it for identifying a eukaryotic transcriptional activator AmyR, which is known to regulate amylolytic gene expression in Aspergillus species. Segmented parts of the binding site of AmyR were randomized to make a DNA library on beads, onto which MalE-tagged AmyR protein and fluorescent anti-MalE tag antibody were bound, followed by selection with a flow cytometer. From a library replacing well-conserved six nucleotides (CGC-CGG) to random ones, the consensus sequence was recovered at a high frequency, demonstrating the reliability of the screening system. Interestingly, similar analysis for another library having randomized intermediate eight nucleotides between the conserved triplets revealed that the selected intermediate sequence had a strong preference for the T nucleotide. Moreover, exactly the same sequence with the upstream region of amyB, a typical AmyR-regulated gene, was found in the selection. These results suggest that this screening system will be a powerful tool for high-throughput analysis of the eukaryotic transcriptome. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.