According to the Frank-Starling relation, cardiac output varies as a function of end-diastolic volume of the ventricle. The cellular basis of the relation is thought to involve length-dependent variations in Ca²⁺ sensitivity of tension; ie, as sarcomere length is increased in cardiac muscle, Ca²⁺ sensitivity of tension also increases. One possible explanation for this effect is that the decrease in myocyte diameter as muscle length is increased reduces the lateral spacing between thick and thin filaments, thereby increasing the likelihood of cross-bridge interaction with actin. To examine this idea, we measured the effects of osmotic compression of single skinned cardiac myocytes on Ca²⁺ sensitivity of tension. Single myocytes from rat enzymatically digested ventricles were attached to a force transducer and piezoelectric translator, and tension-pCa relations were subsequently characterized at short sarcomere length (SL), at the same short SL in the presence of 2.5% dextran, and at long SL. The pCa (−log[Ca²⁺]) for half-maximal tension (ie, pCa₅₀) increased from 5.54±0.09 to 5.65±0.10 (n=7, mean±SD, P<.001) as SL was increased from ≈1.85 to ≈2.25 μm. Osmotic compression of myocytes at short length also increased Ca²⁺ sensitivity of tension, shifting tension-pCa relations to [Ca²⁺] levels similar to those observed at long length (pCa₅₀, 5.68±0.11). These results support the idea that the length dependence of Ca²⁺ sensitivity of tension in cardiac muscle arises in large part from the changes in interfilament lattice spacing that accompany changes in SL.