The length-dependent activation of skeletal myofibrils was examined at the single-sarcomere level with phase-contrast microscopy at sarcomere length (SL) >2.2 microm. At the maximal activation by Ca(2+) (pCa 4.5) the active force linearly decreased with increasing SL, while at partial activation by Ca(2+) (pCa 6.1-6.5) the larger active force was generated at longer SL. Throughout these experiments, the distribution of SL was kept homogeneous upon activation. In addition, we found that the spontaneous oscillation of force and SL frequently occurs in the SL range 2.2-2.6 microm at pCa 6.1-6.2. Either changes in [Ca(2+)] or osmotic compression of the myofilament lattice induced by the addition of dextran T-500, affected both the length dependence of activation and the occurrence of auto-oscillation. These results suggest that the force-generating properties of sarcomeres in striated muscle are determined not only by [Ca(2+)], but also by the lattice spacing as a function of SL.