Two bacterial species with different metabolic features, namely, Pseudomonas aeruginosa and Lactococcus lactis, were used as a comparative experimental model to investigate the antimicrobial target and mechanism of transferrins. In anaerobiosis, P. aeruginosa cells were not susceptible to lactoferrin (hLf) or transferrin (hTf). In aerobiosis, the cells were susceptible but O(2) consumption was not modified, indicating that components of the electron transport chain (ETC) were not targeted. However, the respiratory chain inhibitor piericidin A significantly reduced the killing activity of both proteins. Moreover, 2,6-dichlorophenolindophenol (DCIP), a reducing agent that accepts electrons from the ETC coupled to H(+) extrusion, made P. aeruginosa susceptible to hLf and hTf in anaerobiosis. These results indicated that active cooperation of the cell was indispensable for the antimicrobial effect. For L. lactis cells lacking an ETC, the absence of a detectable transmembrane electrical potential in hLf-treated cells suggested a loss of H(+)-ATPase activity. Furthermore, the inhibition of ATPase activity and H(+) translocation (inverted membrane vesicles) provided direct evidence of the ability of hLf to inhibit H(+)-ATPase in L. lactis. Based on these data, we propose that hLf and hTf also inhibit the H(+)-ATPase of respiring P. aeruginosa cells. Such inhibition thereby interferes with reentry of H(+) from the periplasmic space to the cytoplasm, resulting in perturbation of intracellular pH and the transmembrane proton gradient. Consistent with this hypothesis, periplasmic H(+) accumulation was prevented by anaerobiosis or by piericidin A or was induced by DCIP in anaerobiosis. Collectively, these results indicate that transferrins target H(+)-ATPase and interfere with H(+) translocation, yielding a lethal effect in vitro.