Binding of ATP to the catalytic domain of myosin induces a local conformational change which is believed to cause a major rotation of an 8.5 nm α-helix that is stabilized by the regulatory and essential light chains. Here we attempt to follow this rotation by measuring the mobility and orientation of a fluorescent probe attached near the C- or N-terminus of essential light chain 1 (LC1). Cysteine 178 of wild-type LC1, or Cys engineered near the N-terminus of mutant LC1, was labeled with tetramethylrhodamine and exchanged into skeletal subfragment-1 (S1) or into striated muscle fibers. In the absence of ATP, the fluorescence anisotropy (r) and the rotational correlation time (ρ) of S1 reconstituted with LC1 labeled near the C-terminus were 0.195 and 66.6 ns, respectively. In the presence of ATP, r and ρ increased to 0.233 and 233 ns, indicating considerable immobilization of the probe. A related parameter indicating the degree of order of cross-bridges in muscle fibers, Δr, was small in rigor fibers (-0.009) and increased in relaxed fibers (0,030). For S1 reconstituted with LC1 labeled near the N-terminus, the steady-state anisotropy was 0.168 in rigor, and increased to 0.223 in relaxed state. In fibers, the difference in rigor was large (Δr = 0.080), because of binding to the thin filaments, and decreased to 0.037 in relaxed fibers. These results suggest that before the power stroke, in the presence of ATP or its products of hydrolysis, the termini of LC1 are immobilized and ordered, and after the stroke, they become more mobile and partially disordered. The results are consistent with crystallographic structures that show that the level of putative stabilizing interactions of LC1 with the heavy chain of S1 in the transition state is reduced as the regulatory domain rotates to its post-power stroke position.