Resonance energy transfer probes were attached to skeletal myosin's nucleotide site and regulatory light chain (RLC) to examine nucleotide analog-induced structural transitions. A novel chemical modification of the RLC was developed for specific labeling of the basic N-terminus without affecting myosin ATPase activity. The modification allows attachment of a terbium chelate to rabbit skeletal RLC and was mapped by tryptic digestion to an amino group on the six N-terminal RLC residues. The use of terbium as a resonance energy transfer donor allowed the determination of the efficiency of energy transfer by sensitized emission lifetime measurements that practically eliminate background from unlabeled donor and acceptor sites as well as potential orientation factor artifacts in the calculation of the critical transfer distance. The nucleotide site was labeled with a functional CY3-labeled nucleotide as an energy transfer acceptor. Of the nucleotide states examined, ADP, ADP . vanadate, ADP A1F(4), and ADP . BeFx, the difference between the ADP and ADP . vanadate states was greatest (0.4-nm change), but was not considered to be statistically significant. The binding of actin to ADP-myosin also failed to produce a statistically significant change (0.3-nm change). These results are not consistent with a number of versions of the swinging lever arm hypothesis.