Human-β-defensins 1-3 (HBD-1-3) and their C-terminal analogs Phd-1-3 do not show antibacterial activity against Escherichia coli in the presence of mono- and divalent cations. Activity of peptides was examined against E. coli pretreated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) and salt remedial Escherichia coli ftsEX, a deletion mutant of FtsEX complex [an ATP-binding cassette (ABC) transporter protein], in the presence of Na+, Ca2+, and Mg2+. Activity was observed in the presence of Na+ and Ca2+, although not in the presence of Mg2+ against E. coli, when proton motive force (PMF) was dissipated by CCCP. The peptides exhibited antibacterial activity against E. coli ftsEX even in the presence of Na+ and Ca2+. Our results indicate that HBD-1-3 and Phd-1-3 do not require PMF for their antibacterial activity. The absence of activity against E. coli in the presence of Na+ and Ca2+ ions is due to not only weakened electrostatic interactions with anionic membrane components, but also involvement of electrochemical gradients. However, Mg2+ prevents electrostatic interaction of the peptides with the outer membrane resulting in loss of activity.