Slow skeletal muscle troponin I (ssTnI) expressed predominantly in perinatal heart confers a marked resistance to acidic pH on Ca2+ regulation of cardiac muscle contraction. To explore the molecular mechanism underlying this phenomenon, we investigated the roles of TnI isoforms (ssTnI and cardiac TnI (cTnI)) in the thin filament activation by strongly binding cross-bridges, by exchanging troponin subunits in cardiac permeabilized muscle fibers. Fetal cardiac muscle showed a marked resistance to acidic pH in activation of the thin filament by strongly binding cross-bridges compared to adult muscle, Exchanging ssTnI into adult fibers altered the pH sensitivity from adult to fetal type, indicating that ssTnI also confers a marked resistance to acidic pH on the crossbridge-induced thin filament activation. However, the adult fibers containing ssTnI or cTnI but lacking TnC showed no pH sensitivity. These findings provide the first evidence for the coupling between strongly binding cross-bridges and a pH-sensitive interaction of TnI with TnC in cardiac muscle contraction, as a molecular basis of the mechanism conferring the differential pH sensitivity on Ca2+ regulation.