A polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1, has the potential to be used in soil for the remediation of environmental contamination. It has been found that RHA1 genes, ro06365 (narK) and ro06366, encoding a nitrate/nitrite transporter and nitrite reductase, respectively, were highly upregulated during the growth in sterile soil. In this study, these genes and ro00862, a paralog of ro06366 were characterized to reveal the nitrate and nitrite utilization systems of RHA1. The transcriptional induction of ro06366 (nirB1) and ro00862 (nirB2) by either nitrate or nitrite was revealed by qRT-PCR. Deletion mutants for each gene exhibited retarded growth on either nitrate or nitrite as a sole nitrogen source. Furthermore, their double mutant, Dnit, grew on and consumed neither nitrate nor nitrite as a sole nitrogen source, suggesting that both nirB1 and nirB2 are involved in the utilization of nitrite and nitrate. A narK mutant, DnarK, exhibited no growth on nitrate and retarded growth on nitrite as the sole nitrogen source. DnarK showed no consumption of nitrate and reduced consumption of nitrite, suggesting that narK is essential for nitrate uptake and is partially involved in nitrite uptake. The induced transcription of nirB1, nirB2, and narK was repressed in the presence of 3 mM ammonium or more. The upregulation of nirB1 and narK in sterilized soil containing ammonium and nitrate suggests that the ammonium concentration of the sterilized soil is equivalent to less than 3 mM. The unique nitrogen metabolism system of RHA1 and its importance for the growth in soil are discussed. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.