The two globular Ca2+-binding domains of troponin C are connected by a three-turn, exposed central helix. The requirements of this helical linker for regulatory function are not fully understood. In the present work we investigated the structural requirement of the linker using a series of insertion mutations that differ in predicted flexibility. TnCinrc has a nine-residue flexible random coil insert, TnCinpp has a nine-residue rigid polyproline insert (three turns), and TnCinah has a seven-residue insert with high potential of forming a-helix. TnCinrc and TnCinpp were defective in the activation of the regulated actomyosin ATPase activity in the presence of Ca2+ when compared to wild type or TnCinah, suggesting that altering the flexibility of the central helix impairs the regulatory function of troponin C. TnCinah, TnCinrc, and TnCinpp had 87% ± 3, 62% ± 3, and 58% ± 2 of the wild type activity, respectively (n = 6). All insertions in the central helix resulted in elongation of molecule compared to wild type TnC as determined by Stokes' radius. The Ca2+-affmity, the Ca2+-dependence of the actomyosin ATPase, and the stability of the insertion mutants were similar to wild type. Deletions of up to two turns of the central helix have little effect on troponin C function [Dobrowolski, Z., Xu, G-Q., & Hitchcock-DeGregori, S. E. (1991) J. Biol. Chem. 266, 5703-5710]. In another mutant (TnCd11) the entire central helix, 87KEDAKGKSEEE97, was deleted. With TnCdll, activation of the actomyosin ATPase activity in the presence of Ca2+ was normal, but inhibition in the absence of Ca2+ was less effective. Interaction of TnCdl 1 with Tnl was altered. There was a 2-fold excess of TnCdl 1 in reconstituted Tn complex, consistent with another report [Babu, A., Rao, V. G, Su, H., & Gulati, J. (1993) J. Biol. Chem. 268, 19232-19238]. Our results suggest that the native length and structure of the central helix are optimal for normal regulatory function and that connectivity alone is insufficient for TnC function.