The sigma(54) factor is highly conserved in a large number of bacterial species. From the complete genome sequence of Rhodobacter sphaeroides, it was possible to identify four different sequences encoding potentially functional sigma(54) factors. In this work, we provide evidence that one of these copies (rpoN2) is specifically required to express the flagellar genes in this bacterium. A mutant strain carrying a lesion in the rpoN2 gene was unable to swim even though the RpoN1 and RpoN3 proteins were present in the cytoplasm. The possibility that the different copies of the sigma(54) factor might be specific for the transcription of a particular subset of sigma(54) promoters was reinforced by the fact that a mutant strain carrying a lesion in rpoN1 showed a severe growth defect in nitrogen-free culture medium, even though the rpoN2 and rpoN4 genes were actively transcribed from a plasmid or from the chromosome. Different mechanisms that might be responsible for this specificity are discussed.