Bacterial flagellar protein export requires an ATPase, FliI, and presumptive inhibitor, FliH. We have explored the molecular basis for FliI/ FliH interaction in the human gastric pathogen Helicobacter pylori. By using bioinformatic and biochemical analyses, we showed that residues 1-18 of FliI very likely form an amphipathic alpha-helix upon interaction with FliH, and that residues 21-91 of FliI resemble the N-terminal oligomerization domain of the F-1-ATPase catalytic subunits. A truncated FliI-(2- 91) protein was shown to be folded, although the N-terminal 18 residues were likely unstructured. Deletion and scanning mutagenesis showed that residues 1-18 of FliI were essential for the FliI/ FliH interaction. Scanning mutation of amino acids in the N-terminal 10 residues of FliI indicated that a cluster of hydrophobic residues in this segment was critical for the interaction with FliH. The interaction between FliI and FliH has similarities to the interaction between the N-terminal alpha-helix of the F-1-ATPase alpha-subunit and the globular domain of the F-1-ATPase delta-subunit, respectively. This similarity suggests that FliH may function as a molecular stator.