A time-resolved x-ray diffraction study at a time resolution of 0.53ms was made to investigate the structural origin of latency relaxation (LR) in frog skeletal muscle. Intensity and spacing measurements were made on meridional reflections from the Ca-binding protein troponin and the thick filament and on layer lines from the thin filament. At 16°C, the intensity and spacing of all reflections started to change at 4ms, simultaneously with the LR. At 0°C, the intensity of the troponin reflection and the layer lines from the thin filament and the spacing of the 14.3-nm myosin meridional reflection, but not the spacing of other myosin meridional reflections, began to change at ∼15ms, when the LR also started. Intensity of myosin-based reflections started to change later. When the muscle was stretched to nonoverlap length, the intensity and spacing changes of the myosin reflections disappeared. The simultaneous spacing change of the 14.3-nm myosin meridional reflection with the LR suggests that detachment of myosin heads that are bound to actin in the resting muscle is the cause of the LR.