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 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 54-dependent transcription. Hence, all 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 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 54, and in the 54-activator gene flgR, showed reciprocally reduced transcription of genes that were overexpressed in the fliK mutant. The fliA (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.