The association of a ligand with its cognate cell surface receptor can be facilitated by interactions between the ligand and the lipid phase of the cell membrane. With respect to the N-methyl-D-aspartate receptor (NMDAR), we have previously established a low affinity, nonreceptor-mediated interaction of the peptidic conantokins with synaptic membranes in conjunction with a high affinity binding to the NMDARs present therein [Klein, R. C., Prorok, M., and Castellino, F. J. (2003) J. Pept. Res. 61, 307-317]. In the current study, several techniques including size-exclusion chromatography, circular dichroism, fluorescence, and NMR spectroscopies were used to investigate the binding, conformation, and orientation of conantokins and their variants to a variety of phospholipid (PL) vesicles and multilayers. We have found that conantokins bind to PLs and that the effectors Ca(2+) and spermine slightly increase this binding ability. The conantokins preserve a high degree of helical conformation when bound to vesicles in the presence of Ca(2+). In the absence of Ca(2+), only conantokin-G (con-G) manifests an increase in conantokin helicity with increasing vesicle concentration. In solution, the conantokins appear to be localized at the headgroup of vesicles and do not insert into the hydrophobic core of the bilayer. On aligned PL films, the helical axis of the conantokins can either reside normal to the membrane surface or partition in a parallel orientation, depending on the nature of the conantokins and the PLs used. These orientation preferences may be conjoined with the biological activities of the conantokins.