Ovotransferrin antimicrobial peptide (OTAP-92) is a cationic fragment of hen ovotransferrin (OTf). OTAP-92 consists of 92 amino acid residues located within the 109-200 sequence of the N-lobe of OTf. This study was aimed to delineate the antimicrobial mechanism of OTAP-92 and to identify its interaction with bacterial membranes. OTAP-92 caused permeation of Escherichia coli outer membrane (OM) to 1-N-phenylnaphthylamine fluorescent probe in a dose-dependent manner. These results suggested that OTAP-92 crossed the bacterial OM by a self-promoted uptake. Cytoplasmic membrane of E. coli was found to be the target for OTAP-92 bactericidal activity, as assayed by the unmasking of cytoplasmic beta-galactosidase due to membrane permeabilization in a kinetic manner. Pretreatment of bacteria with uncoupler, carbonyl cyanide m-chlorophenylhydrazone, markedly enhanced permeation of cytoplasmic membrane, suggesting that the membrane permeation due to OTAP-92 is independent of the transmembrane potential. In an E. coli phospholipid liposome model, it was demonstrated that OTAP-92 has the ability to dissipate the transmembrane electrochemical potential. Intrinsic fluorescence spectra of the two tryptophan residues in OTAP-92, using liposomal membrane, have identified the lipid-binding region as a helix-sheet motif, and suggested an adjacent Ca(2+)-sensitive site within OTAP-92. These data indicated that OTAP-92 possesses a unique structural motif similar to the insect defensins. Further, this cationic antimicrobial peptide is capable of killing Gram-negative bacteria by crossing the OM by a self-promoted uptake and cause damage to the biological function of cytoplasmic membrane.