Atomic models of the myosin motor domain with different bound nucleotides have revealed the open and closed conformations of the switch 2 element [Geeves, M.A. & Holmes, K.C. (1999) Annu. Rev. Biochem.68, 687-728]. The two conformations are in dynamic equilibrium, which is controlled by the bound nucleotide. In the present work we attempted to characterize the flexibility of the motor domain in the open and closed conformations in rabbit skeletal myosin subfragment 1. Three residues (Ser181, Lys553 and Cys707) were labelled with fluorophores and the probes identified three fluorescence resonance energy transfer pairs. The effect of ADP, ADP.BeFx, ADP.AlF4- and ADP.Vi on the conformation of the motor domain was shown by applying temperature-dependent fluorescence resonance energy transfer methods. The 50 kDa lower domain was found to maintain substantial rigidity in both the open and closed conformations to provide the structural basis of the interaction of myosin with actin. The flexibility of the 50 kDa upper domain was high in the open conformation and further increased in the closed conformation. The converter region of subfragment 1 became more rigid during the open-to-closed transition, the conformational change of which can provide the mechanical basis of the energy transduction from the nucleotide-binding pocket to the light-chain-binding domain.