Bacillus subtilis can use ammonium and various amino acids as sole nitrogen sources. The utilization of arginine or ornithine is abolished in a sigma L-deficient strain of B. subtilis, indicating that one or several genes involved in this pathway are transcribed by a sigma L-RNA polymerase holoenzyme. Three B. subtilis genes, called rocA, rocB, and rocC, which seem to form an operon, were found near the sacTPA locus (P. Glaser, F. Kunst, M. Arnaud, M.-P. Coudart, W. Gonzales, M.-F. Hullo, M. Ionescu, B. Lubochinsky, L. Marcelino, I. Moszer, E. Presecan, M. Santana, E. Schneider, J. Schweizer, A. Vertes, G. Rapport, and A. Danchin, Mol. Microbiol. 10:371-384, 1993). The expression of this putative operon is induced by arginine and is sigma L dependent. Mutants impaired in the transcription of rocA were obtained. One of these mutants was used as recipient to clone and sequence a new regulatory gene, called rocR. This gene encodes a polypeptide of 52 kDa which belongs to the NtrC/NifA family of transcriptional activators. Upstream activating sequences highly similar to those of NtrC in Escherichia coli were also identified upstream from the rocABC genes. A B. subtilis strain containing a rocR null mutation is unable to use arginine as the sole nitrogen source, indicating that RocR is a positive regulator of arginine catabolism. After LevR, RocR is the second example of an activator stimulating sigma 54-dependent promoters in gram-positive bacteria.