Cecropin A (CA), a bioactive peptide, produced significant lethality to Pantoea agglomerans (PA) at low concentrations. Significant mortality occurred immediately after addition of CA. Separate preincubations of lipopolysaccharides (LPS) from the following bacteria: PA, Serratia marcescens, Escherichia coli (EC), and Salmonella typhimurium with CA were performed prior to the bioassay. CA was also preincubated with diphosphoryl lipid A (DPL-A) from EC and S. minnesota (SM), trilinolein, palmitic, lauric and myristic acids (fatty acids contained in the lipid A of PA-LPS) and bovine brain gangliosides. Spectral analysis to determine the interaction between glycosphingolipids (sphingomyelin, bovine brain gangliosides, and galactocerebrosides) and CA were performed. Results showed that all types of LPS and DPL-A as well as gangliosides studied blocked CA lethality to PA. The level of inhibition of CA antibacterial properties was dependent on LPS and DPL-A concentration. The individual fatty acids and trilinolein did not affect CA lethality to PA. Spectral studies showed complexation between CA and PA-LPS, both types of DPL-A, and the glycosphingolipids. Biological and chemical analyses confirm that CA binds to the diphosphoryl lipid A moiety of LPS.