The effects of calmodulin (CaM) antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and its derivatives or trifluoperazine (TFP) on the Ca2+ binding to CaM were investigated. In the presence of these CaM antagonists, the extent of the Ca2+ binding to CaM increased. Stoichiometrical examination showed that these CaM antagonists dose-dependently increased the sensitivity of all four Ca2+ binding sites on CaM. An analysis of CaM by UV spectroscopy revealed that the conformation of the Ca2+ CaM complex in the presence and absence of these CaM antagonists differed. Therefore, binding of this CaM antagonist to CaM increase the binding of Ca2+ to this protein.