AbstractTroponin I (TnI) peptides (TnI inhibitory peptide residues 104–115, Ip; TnI regulatory peptide resides 1–30, TnI1–30), recombinant Troponin C (TnC) and Troponin I mutants were used to study the structural and functional relationship between TnI and TnC. Our results reveal that an intact central D/E helix in TnC is required to maintain the ability of TnC to release the TnI inhibition of the acto‐S1‐TM ATPase activity. Ca2+‐titration of the TnC‐TnI1–30 complex was monitored by circular dichroism. The results show that binding of TnI1–30 to TnC caused a three‐folded increase in Ca2+ affinity in the high affinity sites (III and IV) of TnC. Gel electrophoresis and high performance liquid chromatography (HPLC) studies demonstrate that the sequences of the N‐ and C‐terminal regions of TnI interact in an anti‐parellel fashion with the corresponding N‐ and C‐domain of TnC. Our results also indicate that the N‐ and C‐terminal domains of TnI which flank the TnI inhibitory region (residues 104 to 115) play a vital role in modulating the Ca2+‐ sensitive release of the TnI inhibitory region by TnC within the muscle filament. A modified schematic diagram of the TnC/TnI interaction is proposed. © 2001 Wiley‐Liss, Inc.