The planctomycetes are a phylogenetically distinct group of bacteria, widespread in aquatic and terrestrial environments. Their cell walls lack peptidoglycan and their compartmentalised cells undergo a yeast-like budding cell division process. Many bacteria regulate a subset of their genes by an enhancer-dependent mechanism involving the alternative sigma factor sigma54 (RpoN, sigmaN) in association with sigma54-dependent transcriptional activators known as enhancer-binding proteins (EBPs). The sigma54-dependent regulon has previously been studied in several groups of bacteria, but not in the planctomycetes. We wished to exploit the recently published complete genome sequence of Pirellula species strain 1 to predict and analyse the sigma54-dependent regulon in this interesting group of bacteria. The genome of Pirellula species strain 1 encodes one homologue of sigma54, and 16 sigma54-dependent EBPs, including 10 two-component response regulators and a homologue of Escherichia coli RtcR. Two EBPs contain forkhead-associated domains, representing a novel protein domain combination not previously observed in bacterial EBPs and suggesting a novel link between the enhancer-dependent regulon and 'eukaryotic-like' protein phosphorylation in bacterial signal transduction. We identified several potential sigma54-dependent promoters upstream of genes and operons including two homologues of csrA, which encodes the global regulator CsrA, and rtcBA, encoding a RNA 3'-terminal phosphate cyclase. Phylogenetic analysis of EBP sequences from a wide range of bacterial taxa suggested that planctomycete EBPs fall into several distinct clades. Also the phylogeny of the sigma54 factors is broadly consistent with that of the host organisms. These results are consistent with a very ancient origin of sigma54 within the bacterial lineage. The repertoire of functions predicted to be under the control of the sigma54-dependent regulon in Pirellula shares some similarities (e.g. rtcBA) as well as exhibiting differences with that in other taxonomic groups of bacteria, reinforcing the evolutionarily dynamic nature of this regulon.