BACKGROUND: The sigma(54) subunit controls a unique class of promoters in bacteria. Such promoters, without exception, require enhancer binding proteins (EBPs) for transcription initiation. Desulfovibrio vulgaris Hildenborough, a model bacterium for sulfate reduction studies, has a high number of EBPs, more than most sequenced bacteria. The cellular processes regulated by many of these EBPs remain unknown. RESULTS: To characterize the sigma(54)-dependent regulome of D. vulgaris Hildenborough, we identified EBP binding motifs and regulated genes by a combination of computational and experimental techniques. These predictions were supported by our reconstruction of sigma(54)-dependent promoters by comparative genomics. We reassessed and refined the results of earlier studies on regulation in D. vulgaris Hildenborough and consolidated them with our new findings. It allowed us to reconstruct the sigma(54) regulome in D. vulgaris Hildenborough. This regulome includes 36 regulons that consist of 201 coding genes and 4 non-coding RNAs, and is involved in nitrogen, carbon and energy metabolism, regulation, transmembrane transport and various extracellular functions. To the best of our knowledge, this is the first report of direct regulation of alanine dehydrogenase, pyruvate metabolism genes and type III secretion system by sigma(54)-dependent regulators. CONCLUSIONS: The sigma(54)-dependent regulome is an important component of transcriptional regulatory network in D. vulgaris Hildenborough and related free-living Deltaproteobacteria. Our study provides a representative collection of sigma(54)-dependent regulons that can be used for regulation prediction in Deltaproteobacteria and other taxa.