Background: In Bacillus subtilis, two major transcriptional factors, GlnR and TnrA, are involved in a sophisticated network of adaptive responses to nitrogen availability. GlnR was reported to repress the transcription of the glnRA, tnrA and ureABC operons under conditions of excess nitrogen. As GlnR and TnrA regulators share the same DNA binding motifs, a genome-wide mapping of in vivo GlnR-binding sites was still needed to clearly defne the set of GlnR/TnrA motifs directly bound by GlnR. Methods: We used chromatin immunoprecipitation coupled with hybridization to DNA tiling arrays (ChIP-on-chip) to identify the GlnR DNA-binding sites, in vivo, at the genome scale. Results: We provide evidence that GlnR binds reproducibly to 61 regions on the chromosome. Among those, 20 regions overlap the previously defned in vivo TnrA-binding sites. In combination with real-time in vivo transcriptional profling using frefy luciferase, we identifed the alsT gene as a new member of the GlnR regulon. Additionally, we characterized the GlnR secondary regulon, which is composed of promoter regions harboring a GlnR/TnrA box and bound by GlnR in vivo. However, the growth conditions revealing a GlnR-dependent regulation for this second category of genes are still unknown. Conclusions: Our fndings show an extended overlap between the GlnR and TnrA in vivo binding sites. This could allow efcient and fne tuning of gene expression in response to nitrogen availability. GlnR appears to be part of complex transcriptional regulatory networks, which involves interactions between diferent regulatory proteins.