Fluorescence and fluorescence anisotropy studies were applied in analyzing the structure of two related octadecapeptides which had been shown to form alpha-helical hexamers. Peptide W, EQLLKALEWLLKELLEKL, exhibits a fluorescence maximum of 345 nm in a phosphate buffer in concentration ranging from 2.8 to 228.7 muM. However, its rotational relaxation time increases from 0.93 ns in a 2.8 muM solution to 2.37 ns in a 228.7 muM solution. The self-association of this peptide is analyzed according to the hydrodynamic theory. The results are in agreement with our previous observations that the peptide forms a hexamer at higher peptide concentrations. Peptide F, EQLLKALEFLLKELLEKL, forms a metastable complex with terphenyl in the phosphate buffer. The complex exhibits a rotational relaxation time of 4.12 ns immediately following the sample preparation, but terphenyl begins to precipitate out of the mixture within a few hours. The dissociation is essentially complete after 2 days. The implication of these results on the structure of hexameric helical peptides is discussed.