The temperature dependence of sliding velocity, force and the number of cross-bridges was studied on regulated actin filaments (reconstituted thin filaments) when they were placed on heavy meromyosin (HMM) attached to a glass surface. The regulated actin filaments were used because our previous study on muscle fibres demonstrated that the temperature effect was much reduced in the absence of regulatory proteins. A fluorescently labelled thin filament was attached to the gelsolin-coated surface of a polystyrene bead. The bead was trapped by optical tweezers, and HMM–thin filament interaction was performed at 20–35°C to study the temperature dependence of force at the single-molecule level. Our experiments showed that there was a small increase in force with temperature (Q10= 1.43) and sliding velocity (Q10= 1.46). The small increase in force was correlated with the small increase in the number of cross-bridges (Q10= 1.49), and when force was divided by the number of cross-bridges, the result did not depend on the temperature (Q10= 1.03). These results demonstrate that the force each cross-bridge generates is fixed and independent of temperature. Our additional experiments demonstrate that tropomyosin (Tm) in the presence of troponin (Tn) and Ca2+ enhances both force and velocity, and a truncated mutant, Δ23Tm, diminishes force and velocity. These results are consistent with the hypothesis that Tm in the presence of Tn and Ca2+ exerts a positive allosteric effect on actin to make actomyosin linkage more secure so that larger forces can be generated.