The BLR-1 and BLR-2 proteins of Trichoderma atroviride are the Neurospora crassa homologs of white collar-1 and -2, two transcription factors involved in the regulation of genes by blue light. BLR-1 and BLR-2 are essential for photoinduction of phr-1, a photolyase-encoding gene whose promoter exhibits sequences similar to well-characterized light regulatory elements of Neurospora, including the albino proximal element and the light response element (LRE). However, despite the fact that this gene has been extensively used as a blue light induction marker in Trichoderma, the function of these putative regulatory elements has not been proved. The described LRE core in N. crassa comprises two close but variably spaced GATA boxes to which a WC-1/-2 complex binds transiently upon application of a light stimulus. Using 5′ serial deletions of the phr-1 promoter, as well as point mutations of putative LREs, we were able to delimit an ~ 50 bp long region mediating the transcriptional response to blue light. The identified light-responsive region contained five CGATB motifs, three of them displaying opposite polarity to canonical WCC binding sites. Chromatin immunoprecipitation experiments showed that the BLR-2 protein binds along the phr-1 promoter in darkness, whereas the application of a blue light pulse results in decreased BLR-2 binding to the promoter. Our results suggest that BLR-2 and probably BLR-1 are located on the phr-1 promoter in darkness ready to perform their function as transcriptional complex in response to light.