Conantokin-R (con-R), a g-carboxyglutamate-containing 27-residue peptide, is a natural peptide inhibitor of the N-methyl-D-aspartate (NMDA) subtype glutamate receptor. Synthetic analogues of con-R were generated to evaluate the importance of the individual structural elements of this peptide in its NMDA receptor (NMDAR) antagonist activity, measured by inhibition of the spermine-enhanced binding of the NMDAR-specific channel blocker, [3H]MK-801, to rat brain membranes. Progressive C-terminal truncations of the 27-residue peptide revealed stages of severe activity loss. These occurred at con-R[1-11] and con-R[1-7], corresponding to the deletions of Leu12-Pro27 and Met8-Pro27 respectively. A second set of analogues featured single Ala substitutions in the fully active con-R[1-17] fragment. The replacement of Met8 and Leu12 by Ala resulted in approximate 20-fold and 55-fold decreases of inhibitor potency, respectively. In addition to these two residues, the only other positions where a single Ala substitution led to substantial losses (from 11-fold to >1000-fold) of activity were those of the first five N-terminal amino acids. Based on the above findings, the binding epitope of con-R was localized to the N-terminal turn of the helix and other residues on one face along two subsequent turns. This contribution pattern of the side-chains in activity closely resembles the results obtained with another member of this peptide family, con-G. The secondary structure and metal ion binding properties of the con-R variants were also evaluated using circular dichroism spectroscopy. Divalent cation-dependent increases of a-helix content were observed in most analogues. However, analogues with replacement of Gla11 and Gla15, as well as truncation fragments shorter than 15 residues, lost the ability to be stabilized by metal ions. These results confirmed the location of the primary divalent cation binding locus at Gla11 and Gla15. Additional interactions were indicated by the reduced a-helix stability in the Ala analogues of Gla4, Lys7, and Arg14.