A cluster of flagellar genes of Borrelia burgdorferi was identified and sequenced. This cluster comprises an operon, designated the flgK operon, which is initiated by a sigma 70-like promoter. The flgK operon consists of flbF (function unknown), flgK (encoding HAP1), flgL (encoding HAP3) and orfX (function unknown), and maps at 185 kb on the chromosome. In other bacteria, the hook-associated proteins HAP1 and HAP3 connect the flagellar filament to the hook and are required for the last stage of flagellar assembly. Reverse transcriptase-PCR analysis indicated that flbF through to orfX are transcribed as a single mRNA, and primer extension analysis revealed that transcription of the flgK operon is initiated by a sigma 70-like promoter upstream of flbF. Subcloning the flgK promoter element into a promoter probe cat vector revealed that the flgK promoter element had strong activity in both Escherichia coli and Salmonella typhimurium. In addition, when this construct was transformed into a fliA mutant of S. typhimurium which lacked a functional flagellar-specific sigma 28 factor, the flgK promoter was still functional. Based on these results, the promoter element of the flagellin gene (fla, hereafter referred to as flaB) was re-examined. flaB encodes the flagellar filament protein, and a sigma gp33-34-like promoter has been reported to be involved in the transcription of this gene. A transcriptional start point was found 1 bp downstream of the reported start site. The sequence around -10 and -35 are consistent with the presence of a sigma 70-like promoter in addition to the putative sigma gp33-34-like promoter for flaB. In contrast to the flgK promoter element, no activity was detected after subcloning a flaB promoter element into the promoter probe cat vector. Because a sigma 70-like promoter rather than a unique flagellar sigma factor is involved in the later stage of flagellar assembly, the regulation of B. burgdorferi flagellar genes is evidently different from that of other bacteria.