Shinned muscle fibers prepared from fetal rabbit heart (28 days of gestation) showed a marked resistance to acidic pH in the Ca2+ regulation of force generation, compared to the fibers prepared from adult heart. SDS-PAGE and immunoblot analysis showed that the slow skeletal troponin I was predominantly expressed in the fetal cardiac muscle, while the cardiac isoform was predominantly expressed in the adult cardiac muscle. Direct exchange of purified slow skeletal and cardiac troponin I isoforms into these skinned muscle fibers revealed that cardiac troponin I made the Ca2+ regulation of contraction sensitive to acidic pH just as in the adult fibers, whereas slow skeletal troponin I made the Ca2+ regulation of contraction resistant to acidic pH just as in the fetal fibers. These results demonstrate that the troponin I isoform switching accounts fully for the change in the pH dependence of Ca2+ regulation of contraction in developmental cardiac muscle.