The binding of various combinations of chymotryptic troponin T subfragments, troponin I and troponin C to tropomyosin, troponin C and troponin I was examined semiquantitatively by using affinity chromatography. The interaction between troponin T₂ and troponin I intensified the interaction between troponin T₂ (or troponin T) and tropomyosin. When a mixture of troponin T₂ and troponin C was applied to a tropomyosin-Sepharose 4 B column, neither troponin T₂ nor troponin C was retained in the presence of Ca²⁺ ion, while only troponin T₂ was bound in the absence of Ca²⁺ ion. Such a Ca²⁺-dependent effect was not observed with troponin T. Troponin T₂ subfragments, except troponin T₂βIII, were retained by troponin C-Sepharose 4 B in the presence of troponin I, even in the solution containing 1.0M NaCl, in the presence and absence of Ca²⁺ ion. On the basis of these findings, the interactions among troponin components and tropomyosin are discussed.