Structural changes in myosin motors and filaments during relaxation from isometric contraction of single frog muscle fibres (sarcomere length 2.14 mm, 4°C) were investigated by X-ray diffraction. Force declined at a steady rate for ca 400 ms after the last stimulus, at nearly constant sarcomere length. During this phase the intensities of the equatorial and meridional M3 X-ray reflections associated with the radial and axial distributions of myosin motors recovered towards their resting values, consistent with detachment of myosin motors. Stiffness measurements indicated that the decline of the number of attached motors is accompanied by a progressive increase in force per motor. The fine structure of the M3 reflection showed that the motors were displaced towards the start of their working stroke during isometric relaxation. The 1.5% increase in the axial periodicity of the myosin filament associated with muscle activation was not reversed. The final part of force decline that occurs after the 400 ms isometric phase of relaxation is accompanied by large redistribution of sarcomere lengths (chaotic relaxation). At the end of this phase, recovery of the equatorial X-ray intensities was largely complete, but the other X-ray signals recovered more slowly than force. Supported by CNR-INFM, MIUR (Italy), MRC (UK), NIH (USA), EMBO, EMBL, ESRF.