Nitrogen (N) utilization by ectomycorrhizal fungi is an essential aspect of their ecosystem function. N deposition changes both the N pools and the carbon/nitrogen (C/N) ratio of the substrates where ectomycorrhizal fungi are found, and it is important to understand how these changes affect the N forms used by ectomycorrhizal fungi. To overcome the difficulties of studying ectomycorrhizal fungi in situ, we investigated all known N genes in the model fungus, Hebeloma cylindrosporum in a culture study. In addition to studying the regulation of all known N utilization genes, we aimed to understand whether there are gene clusters that undergo similar regulation. Lastly we studied how C/N ratio, N transporter type, and N source affected relative gene expression levels. We grew the D2 strain of H. cylindrosporum on a range of inorganic and organic N sources under low, medium, and high C/N ratios. We found three gene clusters that were regulated in a similar pattern. Lastly, we found C/N ratio, N source and N transporter type all affected gene expression levels. Relative expression levels were highest on the high C/N ratio, BSA and diLeucine N sources, and inorganic N transporters were always expressed at higher levels than organic N transporters. These results suggest that inorganic N sources may always the default preference for H. cylindrosporum, regardless of both the N sources and the C/N ratio of the substrate.