A previously unannotated, putative fliK gene was identified in the Campylobacter jejuni genome based on sequence analysis; deletion mutants in this gene had a 'polyhook' phenotype characteristic of fliK mutants in other genera. The mutants greatly overexpressed the alpha(54)-dependent flagellar hook protein FlgE, to form unusual filamentous structures resembling straight flagella in addition to polyhooks. The genome sequence reveals only one gene predicted to encode an orthologue of the NtrC-family activator required for sigma(54)-dependent transcription. Hence, all sigma(54)-dependent genes in the genome would be overexpressed in the fliK mutant together with flgE. Microarray analysis of genome-wide transcription in the mutant showed increased transcription of a subset of genes, often downstream of sigma(54)-dependent promoters identified by a quality-predictive algorithm applied to the whole genome. Assessment of genome-wide transcription in deletion mutants in rpoN, encoding sigma(54), and in the sigma(54)-activator gene flgR, showed reciprocally reduced transcription of genes that were overexpressed in the fliK mutant. The fliA (sigma(28))-dependent regulon was also analysed. Together the data clearly define the roles of the alternative sigma factors RpoN and FliA in flagellar biogenesis in C. jejuni, and identify additional putative members of their respective regulons.