The C-terminal region of cardiac troponin I (cTnI) is known to be important in cardiac function, as removal of the last 17 C-terminal residues of human cTnI has been associated with myocardial stunning. To investigate the C-terminal region of cTnI, three C-terminal deletion mutations in human cTnI were generated: Δ1 (deletion of residue 210), Δ3 (deletion of residues 208-210), and Δ5 (deletion of residues 206-210). Mammalian two-hybrid studies showed that the interactions between cTnI mutants and cardiac troponin C (cTnC) or cardiac troponin T (cTnT) were impaired in Δ3 and Δ5 mutants when compared to wild-type cTnI. Troponin complexes containing 2-[4'-(iodoacetamido) anilino] naphthalene-6-sulfonic acid (IAANS) labeled cTnC showed that the troponin complex containing cTnI Δ5 had a small increase in Ca(2+) affinity (P < 0.05); while the cTnI Δ1- and Δ3 troponin complexes showed no difference in Ca(2+) affinity when compared to wild-type troponin. In vitro motility assays showed that all truncation mutants had increased Ca(2+) dependent motility relative to wild-type cTnI. These results suggest that the last 5 C-terminal residues of cTnI influence the binding of cTnI with cTnC and cTnT and affect the Ca(2+) dependence of filament sliding, and demonstrate the importance of this region of cTnI.