Changes in inter-filament lattice spacing have been proposed as the mechanism underlying myofilament length-dependent activation. Much of the evidence to support this theory has come from experiments in which high molecular weight compounds, such as dextran, were used to osmotically shrink the myofilament lattice. However, whether inter-filament spacing directly affects myofilament calcium sensitivity (EC₅₀) has not been established. In this study, skinned isolated rat myocardium was osmotically compressed over a wide range (Dextran T500; 0-6%) and EC₅₀ was correlated to both inter-filament spacing and I_1,1/I_1,0 intensity ratio. The latter two parameters were determined by X-ray diffraction in a separate group of skinned muscles. Osmotic compression induced a marked reduction in myofilament lattice spacing, concomitant with increases in both EC₅₀ and I_1,1/I_1,0 intensity ratio. However, inter-filament spacing was not well correlated with EC₅₀ (r²=0.78). A much better and deterministic relationship was observed between EC₅₀ and the I_1,1/I_1,0 intensity ratio (r²=0.99), albeit with a marked discontinuity at low levels of dextran compression. That is, a small amount of external osmotic compression (0.38kPa corresponding to 1% Dextran T500) produced a step-wise increase in the I_1,1/I_1,0 ratio concomitant with a step-wise decrease in EC₅₀; these parameters then remained stable over a wide range of further applied osmotic compression (up to 6% dextran). These findings provide support for a "switch-like" activation mechanism within the cardiac sarcomere that is highly sensitive to changes in external osmotic pressure.