Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine distances between probes attached to the most reactive sulfhydryl (SH1) group on individual myosin "heads." We measured intramolecular and intermolecular interhead distances as well as the distance between one head of heavy meromyosin (HMM) mixed with subfragment-1 (S1) heads attached to F-actin under rigor conditions. The SH1 cysteine was specifically labeled with either a donor (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid) or an acceptor probe (5-iodoacetamidofluorescein). In free solution, the distance between these probes was too large to allow significant FRET, but in the rigor complex with F-actin, intermolecular interhead distances between S1 molecules, HMM molecules, or S1 and HMM were determined to be 6.0-6.3 nm. The radial coordinate of the labels relative to F-actin was 5.0-6.4 nm. However, the intramolecular interhead distance in HMMs in which the two heads were labeled with D and A probes was estimated to be larger. The binding affinity of the second head of HMM(D/A) to F-actin may be reduced because of heterogeneous modification of the SH1 groups, such that the probability of single-head binding is increased.