XlnR is a transcription factor that mediates D-xylose-triggered induction of xylanolytic and cellulolytic genes in Aspergillus. In order to clarify the molecular mechanisms underlying XlnR-mediated induction, Aspergillus oryzae XlnR was fused with the c-myc tag and examined by Western blotting. Phosphate-affinity SDS-PAGE revealed that XlnR was present as a mixture of variously phosphorylated forms in the absence of D-xylose, and that D-xylose triggered additional phosphorylation of the protein. D-Xylose-triggered phosphorylation was a rapid process occurring within 5 min prior to the accumulation of xynG2 mRNA, and removal of D-xylose caused slow dephosphorylation, leading to less-phosphorylated forms. At 30 min after removal, the phosphorylation status was almost identical to that in the absence of D-xylose, and the level of xynG2 mRNA started to decrease. These results indicate that XlnR is highly phosphorylated when it is active in transactivation, implying that D-xylose-triggered reversible phosphorylation controls XlnR activity.