Campylobacter jejuni 81116 has two genes coding for flagellin, flaA and flaB. Fully motile wild-type C. jejuni bacteria express the flaA gene, with no flaB message being detected. A nonmotile flaA flaB+ mutant, R1, produced detectable levels of flagellin B which was incorporated into truncated flagella. After R1 had invaded INT-407 cells, a variant with increased motility, R1-V2, was isolated. R1-V2 produced full-length flagella and an increased amount of flagellin B. Transcriptional analysis showed that R1-V2 contained more flaB mRNA than its parental strain, R1. The flaB gene promoter sequence and primer extension experiments confirmed that transcription of the flaB gene is initiated from a sigma 54 promoter. Neither the promoter sequence nor the coding sequence of flaB had changed in R1-V2. In contrast to R1, R1-V2 no longer produced (truncated) flaA mRNA. The sigma 28 flaA promoter sequence was not changed in R1-V2. We propose that expression of the two flagellin genes in C. jejuni 81116 is regulated at the transcriptional level, in such a way that predominantly one gene at a time is transcribed. We compared the levels of invasiveness of the wild-type strain, R1, and R1-V2 for INT-407 cells. The shift in expression from flaA to flaB occurred not only during invasion assays but also under different conditions in the absence of eukaryotic cells.