AmyR is a transcriptional activator in Aspergillus spp. necessary for induction of the amylolytic enzyme genes. It recognizes 5'-CGGN8CGG-3' conserved in a number of the amylolytic gene promoters, and in addition 5'-CGGAAATTTAA-3' in the A. oryzae alpha-amylase promoter. In this report, interaction of AmyR with the 5'-CGGAAATTTAA-3' type binding site in the Taka-amylase gene (taaG2) promoter was precisely characterized by DNase I footprinting analysis and electrophoretic mobility shift assay in vitro, and also by examination of the in vivo activity of the mutated promoters. The in vitro and in vivo analyses indicated that two AmyR molecules bind cooperatively to the 5'-CGGAAATTTAA-3' sequence by recognizing the CGG triplet at the 5'-end and the AGG triplet just downstream of the sequence.