Truncation and acylation were combined to investigate the broad-spectrum bactericidal and hemolytic peptide S4(1-15). Substitution of up to seven residues with dodecanoic acid (C(12)) gradually led to specific antipseudomonal activity: out of 40 bacterial strains tested in vitro, C(12)-S4(8-15) displayed similar minimal inhibitory concentrations (MICs) as S4(1-15) against Pseudomonas aeruginosa sp. (identical MIC(90)) but was practically inactive against most other bacteria or erythrocytes. Surface plasmon resonance and isothermal titration calorimetry experiments revealed the binding properties of S4(1-15) to be consistent with its nonselective activities, while discriminative activities of C(12)-S4(8-15) correlated with high binding affinity to a membrane containing pseudomonal lipopolysaccharides and with lower affinities to membranes containing nonpseudomonal lipopolysaccharides or cholesterol. Various mechanistic studies failed to detect significant differences in secondary structure, bactericidal kinetics, or ability to perturb the cytoplasmic membrane, pointing to a similar mode of action.