Tropomyosin (Tm) consists of 7 quasiequivalent repeats known as “periods,” and its specific function may be associated with these periods. To test the hypothesis that either period 2 or 3 promotes force generation by inducing a positive allosteric effect on actin, we reconstituted the thin filament with mutant Tm in which either period 2 (

Δ

2Tm) or period 3 (

Δ

3Tm) was deleted. We then studied: isometric tension, stiffness, 6 kinetic constants, and the pCa-tension relationship. N-terminal acetylation of Tm did not cause any differences. The isometric tension in

Δ

2Tm remained unchanged, and was reduced to ~60% in

Δ

3Tm. Although the kinetic constants underwent small changes, the occupancy of strongly attached cross-bridges was not much different. The Hill factor (cooperativity) did not differ significantly between

Δ

2Tm (1.79

\pm

0.19) and the control (1.73

\pm

0.21), or

Δ

3Tm (1.35

\pm

0.22) and the control. In contrast, p

{Ca}_{50}

decreased slightly in

Δ

2Tm (5.11

\pm

0.07), and increased significantly in

Δ

3Tm (5.57

\pm

0.09) compared to the control (5.28

\pm

0.04). These results demonstrate that, when ions are present at physiological concentrations in the muscle fiber system, period 3 (but not period 2) is essential for the positive allosteric effect that enhances the interaction between actin and myosin, and increases isometric force of each cross-bridge.

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