This study on the structure–activity relationship of polymyxin B, a cyclic peptide antibiotic, used sixteen synthetic polymyxin B₃ analogs including alanine scanning analogs to elucidate the contribution of the side chains to antimicrobial activity and lipopolysaccharide (LPS) binding. Of these analogs, [Ala⁵]-polymyxin B₃ showed greatly reduced antimicrobial activity against Escherichia coli (E. coli), Salmonella Typhimurium (S. Typhimurium) and Pseudomonas aeruginosa (P. aeruginosa) with MIC values of 4—16 nmol/ml, suggesting that the Dab (α,γ-diaminobutyric acid) residue at position 5 is the most important residue contributing to bactericidal activity. The antibacterial contribution of Dab when located within the lactam ring (positions 5, 8 and 9) was greater than when located outside the ring (positions 1 and 3). [D-Ala⁶]-, [L-Phe⁶]-, [Ala⁷]-, and [Gly⁷]-polymyxin B₃ analogs retained potent antimicrobial activity, indicating that neither the reduction of hydrophobic character of the D-Phe⁶-Leu⁷ region nor the D-configuration at position 6 is indispensable for antimicrobial activity. LPS binding studies showed that decreased hydrophobicity of the lactam ring had little effect, but the N^γ-amino function of the Dab residues at position 1, 3, 5, 8 and 9 greatly affected LPS binding, with the contribution of Dab⁵ being the most significant.