The transcriptional activator XYR1 is the central regulator that governs cellulolytic and xylanolytic gene expression in Trichoderma reesei. However, despite its biological importance, relatively little is known about its functional binding sequences. In the present study, we investigated the binding characteristics and specific target for XYR1 by using DNase I footprinting analysis and electrophoretic mobility shift assays. We demonstrate that XYR1 can interact not only with the 5'-GGCTAA-3' motif but also with several 5'-GGC(A/T)(3)-3' motifs. In silico analysis revealed that the 5'-GGC(A/T)(3)-3' motifs are widespread as single site in 5'-upstream region of all the XYR1-regulated genes. Furthermore, we defined the important nucleotides within the binding site that contribute to specific interaction with XYR1. Our results suggest that, together with the inverted repeat motifs, the single 5'-GGC(A/T)(4)-3' motifs play important roles as functional XYR1-binding sites in the regulation of cellulase and xylanase gene expression in T. reesei.