Cardiac troponin I (cTnI) has a unique N-terminal extension that plays a role in modifying the calcium regulation of cardiac muscle contraction. Restrictive cleavage of the N-terminal extension of cTnI occurs under stress conditions as a physiological adaptation. Recent studies have shown that in comparison with controls, transgenic mouse cardiac myofibrils containing cTnI lacking the N-terminal extension (cTnI-ND) had a lower sensitivity to calcium activation of ATPase, resulting in enhanced ventricular relaxation and cardiac function. To investigate which step(s) of the ATPase cycle is regulated by the N-terminal extension of cTnI, here we studied the calcium dependence of cardiac myosin II ATPase kinetics in isolated cardiac myofibrils. ATP binding and ADP dissociation rates were measured by using stopped-flow spectrofluorimetry with mant-dATP and mant-dADP, respectively. We found that the second-order mant-dATP binding rate of cTnI-ND mouse cardiac myofibrils was 3-fold faster than that of wild-type myofibrils at low Ca(2+) concentrations. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentration, while the wild-type controls were not significantly affected. These data from experiments using native cardiac myofibrils under physiological conditions indicate that modification of the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase.