Indolicidin is a 13-residue antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils that contains five Trp and three Pro residues. Falla et al. [(1996) J. Biol. Chem. 271, 19298] suggested that indolicidin forms a poly-L-proline II helix based upon the circular dichroism (CD) spectra of a closely related peptide (indolicidin methyl ester). In contrast, we found no evidence of poly-L-proline II helix formation in the CD spectra of native indolicidin in various solvents or when bound to micelles and membranes [Ladokhin et al. (1997) Biophys. J. 72, 794]. We interpreted the spectra as arising from unordered and/or beta-turn structures, but noted a sharp negative band at 227 nm arising from the tryptophan residues that would mask spectral features characteristic of poly-L-proline II helix. We have reexamined this issue by means of CD measurements of native indolicidin and several of its analogues. None of the features characteristic of a poly-L-proline helix (or alpha- or 3(10)-helix) were observed for any of the peptides studied. To eliminate artifacts associated with tryptophan, we synthesized indolicidin-L and indolicidin-F in which all five tryptophans were replaced with leucines or phenylalanines, respectively. The changes in CD spectra of these Trp-free peptides upon transfer into membrane-like environments were found to be consistent with the formation of beta-turns. For the native indolicidin in SDS micelles, temperature increases resulted in a coupled diminution of two sharp bands, a negative one at 227 nm and a positive one at 217 nm. This phenomenon, which is absent in indolicidin-L variants with single Leu-->Trp substitutions, is consistent with exciton splitting produced by the stacking of indole rings. Type VI turns in model peptides in aqueous solution are known to be promoted by stacking interactions between cis-proline and neighboring aromatic residues [Yao et al. (1994) J. Mol. Biol. 243, 754]. Molecular modeling of indolicidin with a -Trp(6)-cis-Pro(7)-Trp(8)- type VIa turn demonstrated the feasibility of this turn conformation and revealed the possibility of an accompanying amphipathic structure. We therefore suggest that turn conformations are the principal structural motif of indolicidin and that these turns greatly enhance membrane activity.