In Azospirillum brasilense the glnB and glnA genes are clustered in an operon regulated by three different promoters: two located upstream of glnB (glnBp1-sigma(70), and glnBp2-sigma(N)) and one as yet unidentified promoter, in the glnBA intergenic region. We have investigated the expression of the glnB gene promoter using glnB-lacZ gene fusions, mutation analysis, heterologous expression and DNA band-shift assays. Deletion of the glnB promoter region showed that NtrC-binding sequences were essential for glnB expression under nitrogen limitation. The A. brasilense NtrC protein activated transcription of glnB-lacZ fusions in the heterologous genetic background of Escherichia coli. Expression of glnB-lacZ fusions in two A. brasilense ntrC mutants differed from that in the wild-type strain. In vitro studies also indicated that the purified NtrC protein from E. coli was able to bind to the glnB promoter region of A. brasilense. Our results show that the NtrC protein activates glnBglnA expression under nitrogen limitation in A. brasilense.