To determine the ability of strong-binding myosin cross-bridges to activate the myocardial thin filament, we examined the Ca2+ dependence of force and cross-bridge interaction kinetics at 15 degrees C in the absence and presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1) in chemically skinned myocardium from adult rats. Relative to control conditions, application of 6 microM NEM-S1 significantly increased Ca2+-independent tension, measured at pCa 9.0, from 0.8 +/- 0.3 to 3.7 +/- 0.8 mN mm-2. Furthermore, NEM-S1 potentiated submaximal Ca2+-activated forces and thereby increased the Ca2+ sensitivity of force, i.e. the [Ca2+] required for half-maximal activation (pCa50) increased from pCa 5.85 +/- 0.05 to 5.95 +/- 0.04 (change in pCa50 (dpCa50) = 0.11 +/- 0.02). The augmentation of submaximal force by NEM-S1 was accompanied by a marked reduction in the steepness of the force-pCa relationship for forces less than 0.50 Po (maximum Ca2+-activated force), i.e. the Hill coefficient (n2) decreased from 4.72 +/- 0.38 to 1.54 +/- 0.07. In the absence of NEM-S1, the rate of force redevelopment (ktr) was found to increase from 1.11 +/- 0.21 s-1 at submaximal [Ca2+] (pCa 6.0) to 9.28 +/- 0.41 s-1 during maximal Ca2+ activation (pCa 4.5). Addition of NEM-S1 reduced the Ca2+ dependence of ktr by eliciting maximal values at low levels of Ca2+, i.e. ktr was 9.38 +/- 0.30 s-1 at pCa 6.6 compared to 9.23 +/- 0.27 s-1 at pCa 4. At intermediate levels of Ca2+, ktr was less than maximal but was still greater than values obtained at the same pCa in the absence of NEM-S1. NEM-S1 dramatically reduced both the extent and rate of relaxation from steady-state submaximal force following flash photolysis of the caged Ca2+ chelator diazo-2. These data demonstrate that strongly bound myosin cross-bridges increase the level of thin filament activation in myocardium, which is manifested by an increase in the rate of cross-bridge attachment, potentiation of force at low levels of free Ca2+, and slowed rates of relaxation.