Brevinin-2 related peptide (B2RP; GIWDTIKSMG(10)KVFAGKILQN(20)L.NH(2)), first isolated from skin secretions of the mink frog Lithobates septentrionalis, shows broad-spectrum antimicrobial activity but its therapeutic potential is limited by moderate hemolytic activity. The peptide adopts an alpha-helical conformation in a membrane-mimetic solvent but amphipathicity is low. Increasing amphipathicity together with hydrophobicity by the substitutions Lys(16)-->Leu and Lys(16)-->Ala increased hemolytic activity approximately fivefold without increasing antimicrobial potency. The substitution Leu(18)-->Lys increased both cationicity and amphipathicity but produced decreases in both antimicrobial potency and hemolytic activity. In contrast, increasing cationicity of B2RP without changing amphipathicity by the substitution Asp(4)-->Lys resulted in a fourfold increase in potency against Escherichia coli [minimal inhibitory concentration (MIC) = 6 microm) and twofold increases in potency against Staphylococcus aureus (MIC = 12.5 microm) and Candida albicans (MIC = 6 microm) without changing significantly hemolytic activity against human erythrocytes (LC(50) = 95 microm). The emergence of antibiotic-resistant strains of the Gram-negative bacterium Acinetobacter baumannii constitutes a serious risk to public health. B2RP (MIC = 3-6 microm) and [Lys(4)]B2RP (MIC = 1.5-3 microm) potently inhibited the growth of nosocomial isolates of multidrug-resistant Acinetobacter baumannii. Although the analogs [Lys(4), Lys(18)]B2RP and [Lys(4), Ala(16), Lys(18)]B2RP showed reduced potency against Staphylococcus aureus, they retained activity against Acinetobacter baumannii (MIC = 3-6 microm) and had very low hemolytic activity (LC(50) > 200 microm).