The ¯uorescence resonance energy transfer parameter, f, is de®ned as the e ciency of the energy transfer normalized by the quantum yield of the donor in the presence of acceptor. It is possible to characterize the ¯exibility of the protein matrix between the appropriate ¯uorescent probes by monitoring the temperature dependence of f. The intermonomer ¯exibility of the Ca-actin and Mg-actin ®laments was characterized by using this method at pH values of and .