Flagellar hooks were purified from Helicobacter pylori and Helicobacter mustelae. The 70 x 16 nm H. pylori hook was composed of FlgE subunits of 78 kDa, while the 72 x 16 nm H. mustelae hook was composed of 87 kDa subunits. N-terminal sequence was obtained for the FlgE proteins of both species, and for an internal H. mustelae FlgE peptide. Degenerate oligonucleotide primers allowed amplification of a 1.2 kb fragment from the H. mustelae chromosome, which carried part of the flgE gene. The corresponding H. pylori gene was cloned by immunoscreening of a genomic library constructed in lambda ZAP Express. The translated H. pylori flgE sequence indicated a protein with limited homology with the hook proteins from Salmonella typhimurium and Treponema phagedenis. Mutants of H. pylori and H. mustelae defective in hook production generated by allele replacement were nonmotile and devoid of flagellar filaments but produced both flagellin subunits, which were localized in the soluble fraction of the cell. The level of flagellin production was unchanged in the mutants, indicating that the regulation of flagellin expression in Helicobacter differs from that in the Enterobacteriaceae.