The macrolide antibiotic bafilomycin and the related synthetic compound SB 242784 are potent inhibitors of the vacuolar H+ -ATPases (V-ATPase). It is currently believed that the site of action of these inhibitors is located on the membrane bound c-subunits of V-ATPases. To address the identification of the critical inhibitors binding domain, their specific binding to a synthetic peptide corresponding to the putative 4th transmembrane segment of the c-subunit was investigated using fluorescence resonance energy transfer (FRET), and for this purpose a specific formalism was derived. Another peptide of the corresponding domain of the c' isoform, was checked for binding of bafilomycin, since it is not clear if V-ATPase inhibition can also be achieved by interaction of the inhibitor with the c'-subunit. It was concluded that bafilomycin binds to the selected peptides, whereas SB 242784 was unable to interact, and in addition for bafilomycin, its interaction with the peptides either corresponding to the c- or the c'-subunit isoforms is identical. Since the observed interactions are however much weaker as compared to the very efficient binding of both bafilomycin and SB 242784 to the whole protein, it can be concluded that assembly of all V-ATPase transmembrane segments is required for an efficient interaction.