To explore the functional relationship between the polymerization site a and the nearby high affinity calcium binding site, we analyzed four variant fibrinogens with substitutions at these sites: gamma D364A in the a site and gamma D318A, gamma D320A, and gamma D318 + gamma D320A in the Ca2+ site. In all cases fibrinopeptide A release was normal and thrombin catalyzed polymerization was markedly impaired (unpublished observations). We examined the functional connection between the Ca2+ site and the a site by testing for plasmin protection in the presence of Ca2+ or the a site peptide ligand GPRP. SDS-PAGE analysis of the products showed that gamma D364A fibrinogen was protected from plasmin cleavage by Ca2+ but not by the GPRP peptide. In contrast, neither Ca2+ nor the GPRP peptide protected gamma D318A, gamma D320A, or gamma D318 + gamma D320A fibrinogens from complete plasmin cleavage. These results suggest that the structural integrity of the calcium binding site is required for expression of the a site. In contrast, the structural integrity of the a site has no functional consequence on Ca2+ binding to this high affinity site.