The natural antimicrobial cationic peptide protegrin-1 displays a broad spectrum of antimicrobial activity and rapidly kills pathogens by interacting with their cell membrane. We investigated the structure-activity relationships of three protegrin-1 analogues: IB-367 (RGGLCYCRGRFCVCVGR-NH(2)), BM-1 (RGLCYCRGRFCVCVG-NH(2)) and BM-2 (RGLCYRPRFVCVG-NH(2)). Our antimicrobial and antifungal activity studies of these peptides showed that BM-1 was much more active than IB-367 against Gram-positive bacteria and fungi, whereas BM-2 was inactive. The BM-1 peptide showed fourfold reduced haemolysis relative to IB-367, an additional advantage of this peptide. In addition, BM-1 was about 15% cheaper than IB-367 to synthesize. The absence of two cysteine residues in the BM-2 sequence could be the main reason for its unstable conformation and antimicrobial inactivity. The solution structures of these peptides were determined in dimethyl sulphoxide using two-dimensional NMR and restrained molecular dynamics calculations. IB-367 and BM-1 formed short, antiparallel, beta-hairpin structures connected by a type II' beta-turn. The shorter, inactive BM-2 analogue exhibited major and minor conformations (predominantly unordered) in the NMR spectra and was much more flexible.