We tested the hypotheses that Ca(2+) concentration ([Ca(2+)]) and sarcomere length (SL) modulate force development via graded effects on cross-bridge kinetics in chemically permeabilized rat cardiac trabeculae. Using sinusoidal length perturbations, we derived the transfer functions of stiffness over a range of [Ca(2+)] at a constant SL of 2.1 micrometer (n = 8) and at SL of 2.0, 2.1, and 2.2 micrometer (n = 4). We found that changes in SL affected only the magnitude of stiffness, whereas [Ca(2+)] affected the magnitude and phase-frequency relations. The data were fit to complex functions of two exponential processes. The characteristic frequencies (b and c) of these processes are indexes of cross-bridge kinetics, with b relating to cross-bridge attachment to and c to detachment from certain non-force-generating states. Both were significantly affected by [Ca(2+)], with an increase in b and c of 140 and 44%, respectively, over the range of [Ca(2+)] studied (P < 0.01). In contrast, SL had no effect on the characteristic frequencies (P > 0.6). We conclude that Ca(2+) activation modulates force development in rat myocardium, at least in part, via a graded effect on cross-bridge kinetics, whereas SL effects are mediated mainly by recruitment of cross bridges.