1. A method for isolation of troponin from native tropomyosin was described. 2. Troponin in combination with tropomyosin restored the whole activity of native tropomyosin in sensitizing the interaction of myosin and actin to Ca ion. 3. Troponin was found to bind nearly 4 moles of Ca per 105 g, of which most were exchangeable. The result of the experiment to determine the binding constant of these Ca binding sites was explained by assuming that half of the binding sites possessed a binding constant of 1.3 × 106 M−1and the remaining half 5×104 M−1. 4. The amount of exchangeable Ca in the contractile system was mainly accounted for by the Ca-binding capacity of troponin, which was not influenced by other contractile proteins or ATP. 5. Cardiac troponin showed a much higher affinity for Sr ion than skeletal troponin. The ratio of the former affinity to the latter was in good agreement with the ratio of the sensitivity to Sr ion of a reconstituted contractile system containing cardiac troponin to that containing skeletal troponin. Based on these findings and the results described above, it was concluded that the sensitivity of a contractile system to Ca ion is solely dependent upon the affinity for Ca ion of the troponin molecule present. 6. The mechanism of troponin regulation of the interaction of actin and myosin was discussed.