Microorganisms have evolved transcriptional networks to prioritize utilization of available nutrient sources. For filamentous fungi, such as Neurospora crassa, this entails distinguishing between a variety of organic and inorganic nitrogen sources. Here, we transcriptionally profiled the response of N. crassa to a variety of nitrogen sources and used DNA affinity purification sequencing to characterize the role of regulatory genes and their direct downstream targets. We identified a transcription factor responsible for regulating genes involved in amino acid and mannose metabolism. By comparing the genes regulated by transcription factors that regulate specific nitrogen utilization pathways and transcription factors that regulate utilization of all nitrogen sources that require metabolic processing before utilization, we revealed aspects of the nitrogen regulatory network.Sensing available nutrients and efficiently utilizing them is a challenge common to all organisms. The model filamentous fungus Neurospora crassa is capable of utilizing a variety of inorganic and organic nitrogen sources. Nitrogen utilization in N. crassa is regulated by a network of pathway-specific transcription factors that activate genes necessary to utilize specific nitrogen sources in combination with nitrogen catabolite repression regulatory proteins. We identified an uncharacterized pathway-specific transcription factor, amn-1, that is required for utilization of the nonpreferred nitrogen sources proline, branched-chain amino acids, and aromatic amino acids. AMN-1 also plays a role in regulating genes involved in responding to the simple sugar mannose, suggesting an integration of nitrogen and carbon metabolism. The utilization of nonpreferred nitrogen sources, which require metabolic processing before being used as a nitrogen source, is also regulated by the nitrogen catabolite regulator NIT-2. Using RNA sequencing combined with DNA affinity purification sequencing, we performed a survey of the role of NIT-2 and the pathway-specific transcription factors NIT-4 and AMN-1 in directly regulating genes involved in nitrogen utilization. Although previous studies suggested promoter binding by both a pathway-specific transcription factor and NIT-2 may be necessary for activation of nitrogen-responsive genes, our data show that pathway-specific transcription factors regulate genes involved in the catabolism of specific nitrogen sources, while NIT-2 regulates genes involved in utilization of all nonpreferred nitrogen sources, such as nitrogen transporters. Together, these transcription factors form a nutrient sensing network that allows N. crassa cells to regulate nitrogen utilization.RNAseq data used in this study were deposited in the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) and are accessible through GEO series accession number GSE150256 (68). Processed RNAseq data are available in Dataset S1. DAPseq data used in this study were deposited in the NCBI Sequence Read Archive (SRA) and are accessible through SRA series accession number PRJNA436200 (69). Processed DAPseq data are available in Dataset S3. The numerical values used to generate all mycelial dry weight and qRT-PCR graphs are shown in Dataset S2. Strains constructed in this study are available from the Fungal Genetics Stock Center (www.fgsc.net).