The role of tropomyosin (Tm) in the elementary steps of the cross-bridge cycle in bovine myocardium was investigated. The thin filament was selectively removed using gelsolin (thin filament severing protein), and the actin filament was reconstituted from G-actin. Tm was further reconstituted without troponin (Tn), and the kinetic constants of the elementary steps of the cross-bridge cycle were deduced using sinusoidal analysis at pCa 4.66, pH 7.00, and 25°C. The association constant of MgATP to cross-bridges (K1) after reconstitution of Tm was 20.7 ± 2.3 mM-1, which was about 2' of the control (untreated) myocardium (9.1±1.3 mM-1). By the reconstitution of Tm, the equilibrium constant of the cross-bridge detachment step (K2), the phosphate (Pi) association constant (K5) and the equilibrium constant of the force-generation step (K4), which significantly changed in the actin filament-reconstituted myocardium, recovered to those of the control myocardium. Active tension after reconstitution of Tm was 0.69' of the control myocardium, a value between the control (1') and the actin filament-reconstituted myocardium (0.59'). Tm-reconstituted myocardium was further reconstituted with Tn, and the effect of MgATP on the rate constants (K1, K2) was studied. By reconstitution with Tn, the myocardium regained the Ca2+-sensitivity and the active tension became 0.83' of the control myocardium. In addition, K1 recovered the value of the control myocardium with the Tn reconstitution. These results indicate that both Tm and Tn enhance the force generated by each cross-bridge, and that Tm is primarily responsible for the change in kinetic constants of elementary steps of the cross-bridge cycle.