The pathway of cell membrane lysis by the peptide antibiotic cecropin B (CB), which contains both a hydrophobic and an amphipathic alpha-helix, was analysed by assessing the morphological changes of Escherichia coli following treatment with the peptide. Exposure of green fluorescent protein (GFP)-expressing E. coli to CB does not lead to an efflux of GFP. Moreover, transmission electron microscopic (TEM) examination of cecropin B-treated cells showed that severe swelling precedes cell death and that the outer membrane becomes distended away from the plasma membrane. Using immuno-gold staining and TEM of E. coli expressing the maltose-binding protein in the cytoplasm, it was apparent that the protein remains restricted to the cytoplasmic compartment. These observations suggest that CB causes gross disruption of the outer membrane of Gram-negative bacteria. Circular dichroism measurements of CB in the presence of cell membrane-mimicking liposomes showed that CB forms secondary structure dependent on the ratio of [lipid]/[peptide]. These observations from this study are important for the future design of custom antimicrobial peptides.