The importance of the metabolic route of nitrogen in the fungus Penicillium rubens (strain PO212) is studied in relation to its biocontrol activity (BA). PO212 can resist a high concentration of chlorate anion and displays a classical nitrate-deficiency (nit-) phenotype resulting in poor colonial growth when nitrate is used as the main source of nitrogen. Analyses of genes implicated in nitrate assimilation evidenced the strong sequence conservation of PO212 and CH8 genome with penicillin producers such as reference strain P. rubens Wisconsin 54-1255, P2niaD18 and Pc3, however also revealed the presence of mutations. PO212 carries a mutation in the gene coding for zinc-binuclear cluster transcription factor NirA that specifically mediates the regulation of genes involved in nitrate assimilation. The nirA1 mutation causes an early stop of NirA factor, losing 66% of its sequence. The NirA1 mutant form is unable to mediate a nitrate-dependent regulation of nitrate and nitrite reductase coding genes. In this study, we study another isolate, CH8, with potential BA and nit- phenotype. A mutation in the nitrate permease coding gene crnA was found in CH8. An insertion of a guanine in the coding sequence cause a frameshift in CrnA with the loss of the last two transmembrane domains. Analysis of PO212 and CH8 isolates and complementation strains show the importance of NirA regulator in maintaining correct transcriptional levels of nitrate and nitrite reductases and suggest CrnA as the main nitrate transporter. the presence of alternative transporter for chlorate and the existence of a mechanism for preventing nitrite derived toxicity in Penicillum. BA of PO212 is partially altered when nirA1 mutation was complemented. This result and the finding of CH8, a novel biocontrol P. rubens strain with a nit- phenotype, suggest that nitrogen metabolism is a component of biocontrol capacity.