Fluorescence resonance energy transfer measurements have been made between Cys-374 on actin and Cys-177 on the alkali light chain of myosin subfragment 1 (S1) using several pairs of donor-acceptor chromophores. The labeled light chain was exchanged into subfragment 1 and the resulting fluorescently labeled subfragment 1 isolated by ion-exchange chromatography on SP-Trisacryl. The efficiency of energy transfer was measured by steady-state fluorescence in a strong binding complex of acto-S1 and found to represent a spatial separation between the two probes of 5.6-6.3 nm. The same measurements were then made with weak binding acto-S1 complexes generated in two ways. First, actin was complexed with p-phenylenedimaleimide-S1, a stable analogue of S1-adenosine 5'-triphosphate (ATP), obtained by cross-linking the SH1 and SH2 heavy-chain thiols of subfragment 1 [Greene, L. E., Chalovich, J. M., & Eisenberg, E. (1986) Biochemistry 25, 704-709]. Large increases in transfer efficiency indicated that the two probes had moved closer together by some 3 nm. Second, weak binding complexes were formed between subfragment 1 and actin in the presence of the regulatory proteins troponin and tropomyosin, the absence of calcium, and the presence of ATP [Chalovich, J. M., & Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437]. The measured efficiency of energy transfer again indicated that the distance between the two labeled sites had moved closer by about 3 nm. These data support the idea that there is a considerable difference in the structure of the acto-S1 complex between the weakly and strongly bound states.