Binding of La3+ to calmodulin (CaM) and its effects on the complexes of CaM and CaMbinding peptide, polistes mastoparan (Mas), were investigated by nuclear magnetic resonance (NMR) spectroscopy, fluorescence and circular dichroism spectroscopy, and by the fluorescence stopped-flow method. The four binding sites of La3+ on CaM were identified as the same as the binding sites of Ca2+ on CaM through NMR titration of La3+ to uniformly 15N-labeled CaM. La3+ showed a slightly higher affinity to the binding sites on the N-terminal domain of CaM than that to the C-terminal. Large differences between the 1H-15N heteronuclear single quantum coherence (HSQC) spectra of Ca4CaM and La4CaM suggest conformational differences between the two complexes. Fluorescence and CD spectra also exhibited structural differences. In the presence of Ca2+ and La3+, a hybrid complex, Ca2La2CaM, was formed, and the binding of La3+ to the N-terminal domain of CaM seemed preferable over binding to the C-terminal domain. Through fluorescence titration, it was shown that La4CaM and Ca2La2CaM had similar affinities to Mas as Ca4CaM. Fluorescence stopped-flow experiments showed that the dissociation rate of La3+ from the C-terminal domain of CaM was higher than that from the N-terminal. However, in the presence of Mas, the dissociation rate of La3+ decreased and the dissociation processes from both global domains were indistinguishable. In addition, compared with the case of Ca4CaM-Mas, the slower dissociations of Mas from La4CaM-Mas and Ca2La2CaM-Mas complexes indicate that in the presence of La3+, the CaM-Mas complex became kinetically inert. A possible role of La3+ in the Ca2+-CaM-dependent pathway is discussed.