(1) Previous mutational analyses have shown that residue π3R398 of the π-subunit is a key residue for binding of the inhibitory antibiotic aurovertin to Escherichia coli F1F0-ATP synthase. Here, we studied purified F1 from the πR398C and πR398W mutants. ATPase activity in both cases was resistant to aurovertin inhibition. The fluorescence spectrum (λexc = 278 or 295 nm) of πR398W F1 showed a significant red-shift as compared to wild-type and πR398C enzymes, indicating that residue πR398 lies in a polar environment. On the basis of this and previous evidence, we propose that aurovertin binding to F1-ATPase involves a specific charged donor-acceptor H-bond between residue πR398 and the 7-hydroxyl group of aurovertin. (2) The fluorescent substrate analog lin-benzo-ADP was shown to bind to πR398W F1 catalytic sites with the same Kd values as to wild-type F1 and with the same quenching of the fluorescence of the analog. Fluorescence energy transfer was seen between the πR398W residue and bound lin-benzo-ADP. Analysis of transfer efficiency at varying stoichiometry of bound lin-benzo-ADP showed that interaction occurred between one /3R398W residue and one catalytic-site-bound analog molecule at a distance of approximately 23 Å. The relationships of the aurovertin and catalytic sites in the primary and tertiary structure are discussed.