The increase in myofilament Ca2+responsiveness on an increase in sarcomere length (SL) is, in part, the cellular basis for Frank-Starling’s law of the heart. It has been suggested that a decrease in myofilament lattice spacing (LS) in response to an increase in SL underlies this phenomenon. This hypothesis is supported by previous studies in which reduced muscle width induced by osmotic compression was associated with an increase in Ca2+sensitivity, mimicking those changes observed with an increase in SL. To evaluate this hypothesis, we directly measured LS by synchrotron x-ray diffraction as function of SL in skinned rat cardiac trabeculae bathed in 0% to 6% dextran solutions (MW 413 000). We found that EC50, [Ca2+] at which force is half-maximal, at SL between 1.95 and 2.25 &mgr;m did not vary in proportion to LS when 3% or 6% dextran solutions were applied. We also found that moderate compression (1% dextran) of skinned trabeculae at SL=2.02 &mgr;m reduced LS (LS=42.29±0.14 nm) to match that of uncompressed fibers at a long SL (SL=2.19 &mgr;m; LS=42.28±0.15 nm). Whereas increasing SL from 2.02 to 2.19 &mgr;m significantly increased Ca2+sensitivity as indexed by the EC50parameter (2.87±0.11 &mgr;mol/L to 2.52±0.12 &mgr;mol/L), similar reduction in myofilament lattice spacing achieved by compression with 1% dextran did not alter Ca2+sensitivity (2.87±0.10 &mgr;mol/L) at the short SL. We conclude that alterations in myofilament lattice spacing may not be the mechanism that underlies the sarcomere length–induced alteration of calcium sensitivity in skinned myocardium.