Interactions among several components of the flagellar export apparatus of Salmonella were studied using affinity chromatography, affinity blotting, and fluorescence resonance energy transfer (FRET). The components examined were two integral membrane proteins, FlhA and FlhB, and two soluble components, FliH and the ATPase FliI. Affinity chromatography and affinity blotting demonstrated a heterologous interaction between FlhA and FlhB but not homologous FlhA-FlhA or FlhB-FlhB interactions. Both FlhA and FlhB consist of N-terminal transmembrane domains and C-terminal cytoplasmic domains (FlhA(C) and FlhB(C)). To study the interactions among the cytoplasmic components (FlhA(C), FlhB(C), FliH, and FliI), FRET measurements were carried out using fluorescein-5-isothiocyanate (FITC) as donor and tetramethylrhodamine-5- (and 6-) isothiocyanate (TRITC) as acceptor. To reveal the nature of the binding interactions, measurements were carried out in physiological buffer, at high salt (0.5 M NaCl) and in 30% 2-propanol. The results indicated that FlhA(C) could bind to FlhB(C) and both FlhA(C) and FlhB(C) could bind to themselves. Both FlhA(C) and FlhB(C) bound to FliH and FliI. Several in-frame deletion mutants of FliH were examined and found to have only minor effects of decreased binding to FlhA(C) and FlhB(C); deletions in the interior of the FliH sequence had a greater effect than those at the N terminus. The FliI mutants examined bound FlhA(C) and FlhB(C) about the same as or slightly more weakly than wild-type FliI. FliH bound more weakly to FliI carrying the N-terminal double mutation R7C/L12P than it did to wild-type FliI, confirming the importance of the N terminus of FliI for its interaction with FliH.