Antimicrobial cationic peptides are prevalent throughout nature as part of the intrinsic defenses of most organisms, and have been proposed as a blueprint for the design of novel antimicrobial agents. They are known to interact with membranes, and it has been frequently proposed that this represents their antibacterial target. To see if this was a general mechanism of action, we studied the interaction, with model membranes and the cytoplasmic membrane of Escherichia coli, of 12 peptides representing all 4 structural classes of antimicrobial peptides. Planar lipid bilayer studies indicated that there was considerable variance in the interactions of the peptides with model phospholipid membranes, but generally both high concentrations of peptide and high transmembrane voltages (usually -180 mV) were required to observe conductance events (channels). The channels observed for most peptides varied widely in magnitude and duration. An assay was developed to measure the interaction with the Escherichia coli cytoplasmic membrane employing the membrane potential sensitive dye 3,5-dipropylthiacarbocyanine in the outer membrane barrier-defective E. coli strain DC2. It was demonstrated that individual peptides varied widely in their ability to depolarize the cytoplasmic membrane potential of E. coli, with certain peptides such as the loop peptide bactenecin and the alpha-helical peptide CP26 being unable to cause depolarization at the minimal inhibitory concentration (MIC), and others like gramicidin S causing maximal depolarization below the MIC. We discuss the mechanism of interaction with the cytoplasmic membrane in terms of the model of Matsuzaki et al. [(1998) Biochemistry 37, 15144-15153] and the possibility that the cytoplasmic membrane is not the target for some or even most cationic antimicrobial peptides.