We have monitored the effects of KLKL(5)KLK (KLK), a derivative of a natural cationic antimicrobial peptide (CAP) on isolated membrane vesicles, and investigated the partition of the peptide within these structures. KLK readily interacted with fluorescent dyes entrapped in the vesicles without apparent pore formation. Fractionation of vesicles revealed KLK predominantly in the membrane. Peptide-treated vesicles appeared with generally disorganized bilayers. While KLK showed no effect on osmotic resistance of human erythrocytes, dramatic decrease in core and surface membrane fluidity was observed in peptide-treated erythrocyte ghosts as measured by fluorescence anisotropy. Finally, CD spectroscopy revealed lipid-induced random coil to beta-sheet and beta-sheet to alpha-helix conformational transitions of KLK. Together with the oligonucleotide oligo-d(IC)(13) [ODN1a], KLK functions as a novel adjuvant, termed IC31. Among other immunological effects, KLK appears to facilitate the uptake and delivery of ODN1a into cellular compartments, but the nature of KLK's interaction with the cell surface and other membrane-bordered compartments remains unknown. Our results suggest a profound membrane interacting property of KLK that might contribute to the immunostimulatory activities of IC31.