Conantokin-G (con-G) is a 17-amino-acid polypeptide that acts as an N-methyl-D-aspartate (NMDA) antagonist. This action has been attributed to a specific but noncompetitive inhibition of the positive modulatory effects of polyamines at NMDA receptors. Con-G possesses several unusual structural features, including five gamma-carboxyglutamate (Gla) residues and a high degree of helicity in aqueous media. Previous structure-activity studies indicated that one or more Gla residues are necessary for NMDA antagonist activity. Con-G analogues were synthesized with alanine (Ala), serine (Ser), and phosphoserine substituted for Gla to assess the contribution of individual Gla residues to biological activity and secondary structure. Replacement of Gla in positions 3 and 4 resulted in polypeptides with markedly reduced and no NMDA antagonist actions, respectively. In contrast, Gla residues in positions 7, 10, and 14 are not required for NMDA antagonist actions because the potencies of con-G analogues containing Ser7, Ser10, Ala14, and Ser14 to inhibit spermine-stimulated [3H]MK-801 binding are similar to the parent peptide. Moreover, the Ala7 derivative of con-G was about fourfold more potent than the parent peptide both as an inhibitor of spermine-stimulated increases in [3H]MK-801 binding (IC50 of approximately 45 nM) and in reducing NMDA-stimulated increases in cyclic GMP levels (IC50 of approximately 77 nM) in cerebellar granule cell cultures. Although con-G and its analogues assumed mixtures of 3(10) and alpha-helices, no clearcut relationship was evinced between the NMDA antagonist properties of these peptides and the degree of helicity they assumed in aqueous solutions. Together with the inability of con-G to affect 5,7-dichloro[3H]kynurenic acid, [3H]CGP-39653, and [3H]ifenprodil binding, these data are consistent with the hypothesis that this polypeptide acts at a unique, polyamine-associated site on NMDA receptors.