The length-dependence of myofilament Ca2+ sensitivity in cardiac muscle appears to be a function of length-dependent variation in the lateral separation of actin and myosin filaments. The goal of this study was to determine how force, Ca2+ sensitivity, and Ca2+ binding to troponin C are correlated in skinned bovine ventricular muscle bundles set at sarcomere length 1.9 microm and subjected to varying degrees of osmotic compression with Dextran T-500. With 5, 10, and 15% Dextran T-500 the muscle diameter was reduced by 13, 21, and 25%, respectively. Addition of 5% Dextran T-500 caused increases in developed force, Ca2+ sensitivity, and in the affinity of Ca2+ for the regulatory binding site on troponin C. All of these parameters were reversed back toward control levels with 10% Dextran T-500. With 15% Dextran T-500 all parameters were decreased to below control levels. These data indicate that (1) there is an optimal filament separation at which both Ca2+ sensitivity and Ca2+ binding are maximized, and (2) Ca2+-troponin C affinity is linked to changes in Ca2+ sensitivity rather than to changes in interfilament spacing.