The simultaneous analysis of muscle force, length, velocity, and time has been shown to precisely characterize the mechanical performance of isolated striated muscle. We tested the hypothesis that the three-dimensional force-velocity-length relationship reflects mechanical properties of sarcomeres. In hamster diaphragm strips, instantaneous sarcomere length (SL) and muscle length were simultaneously measured during afterloaded twitches. SL was measured by means of laser diffraction. We also studied the influence of initial SL, abrupt changes in total load, and 2 x 10-7 M dantrolene. Baseline resting SL at the apex of the length-active tension curve was 2.2 +/- 0.1 microns, whereas SL at peak shortening was 1.6 +/- 0.1 microns in the preloaded twitch and 2.1 +/- 0.1 microns in the "isometric" twitch. Over the whole load continuum and at any given level of isotonic load, there was a unique relationship between instantaneous sarcomere velocity and instantaneous SL. Part of this relationship was time independent and initial SL independent and was markedly downshifted after dantrolene. When five different muscle regions were considered, there were no significant variations of SL and sarcomere kinetics along the muscle. These results indicate that the time- and initial length-independent part of the instantaneous force-velocity-length relationship previously described in muscle strips reflects intrinsic sarcomere mechanical properties.