We performed a genetic analysis of flagellar regulation in Campylobacter jejuni, from which we elucidated key portions of the flagellar transcriptional cascade in this bacterium. For this study, we developed a reporter gene system for C. jejuni involving astA, encoding arylsulphatase, and placed astA under control of the sigma 54-regulated flgDE2 promoter in C. jejuni strain 81-176. The astA reporter fusion combined with transposon mutagenesis allowed us to identify genes in which insertions abolished flgDE2 expression; genes identified were on both the chromosome and the plasmid pVir. Included among the chromosomal genes were genes encoding a putative sensor kinase and the sigma 54-dependent transcriptional activator, FlgR. In addition, we identified specific flagellar genes, including flhA, flhB, fliP, fliR and flhF, that are also required for transcription of flgDE2 and are presumably at the beginning of the C. jejuni flagellar transcriptional cascade. Deletion of any of these genes reduced transcription of both flgDE2 and another sigma 54-dependent flagellar gene, flaB, encoding a minor flagellin. Transcription of the sigma 28-dependent gene flaA, encoding the major flagellin, was largely unaffected in the mutants. Further examination of flaA transcription revealed significant sigma 28-independent transcription and only weak repressive activity of the putative anti-sigma 28 factor FlgM. Our study suggests that sigma 54-dependent transcription of flagellar genes in C. jejuni is linked to the formation of the flagellar secretory apparatus. A key difference in the C. jejuni flagellar transcriptional cascade compared with other bacteria that use sigma 28 for transcription of flagellar genes is that a mechanism to repress significantly sigma 28-dependent transcription of flaA in flagellar assembly mutants is absent in C. jejuni.