Two major xylanases (XYN I and XYN II) of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) are simultaneously expressed during growth on xylan but respond differently to low-molecular-weight inducers. In vivo footprinting analysis of the xylanase1 (xyn1) promoter revealed three different nucleotide sequences (5′-GGCTAAATGCGACATCTTAGCC-3′ [an inverted repeat of GGCTAA spaced by 10 bp], 5′-CCAAT-3′, and 5′-GGGGTCTAGACCCC-3′ [equivalent to a double Cre1 site]) used to bind proteins. Binding to the Cre1 site is only observed under repressed conditions, whereas binding to the two other motifs is constitutive. Applying heterologously expressed components of the H. jecorina cellulase regulators Ace1 and Ace2 and the xylanase regulator Xyr1 suggests that Ace1 and Xyr1 but not Ace2 contact both GGCTAA motifs. H. jecorina transformants containing mutated versions of the xyn1 promoter, leading to elimination of protein binding to the left or the right GGCTAA box revealed either strongly reduced or completely eliminated induction of transcription. Elimination of Cre1 binding to its target released the basal transcriptional level from glucose repression but did not influence the inducibility of xyn1 expression. Mutation of the CCAAT box prevents binding of the Hap2/3/5 complex in vitro and is partially compensating for the loss of transcription caused by the mutation of the right GGCTAA box. Finally, evidence for a competition of Ace1 and Xyr1 for the right GGCTAA box is given. These data prompted us to hypothesize that xyn1 regulation is based on the interplay of Cre1 and Ace1 as a general and specific repressor with Xyr1 as transactivator.