UV resonance Raman spectra of the antimicrobial peptide (AMP) Anoplin (L-Anoplin-NH2) and two of its derivatives (enantiomer D-Anoplin-NH2 and C-terminus deamidated L-Anoplin-OH) were measured in aqueous buffer solution and in membrane-mimetic environments including 2,2,2-trifluoro ethanol (TFE), zwitterionic lipid dipalmitoylglycerophosphocholine (DPPC) and anionic lipid dipalmitoylglycerophosphoglycerol (DPPG) vesicle solutions. All three peptides were found to adopt random-coil/β turn-like conformation in aqueous solution over the temperature range of 1–60 °C. The conformation was found to become more α-helical in membrane-mimetic solutions such as TFE and DPPG but not in DPPC for all Anoplin derivatives. The data demonstrate that Anoplin preferentially binds to the anionic over the zwitterionic model cell membranes. Results also showed that deamidation does not change the conformation of L-Ano-NH2 very significantly, but does alter membrane rupturing and antimicrobial activities thus confirming that it is the physicochemical properties rather than the peptide conformation that define the mechanism of AMP action. Copyright © 2010 John Wiley & Sons, Ltd.