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 4B 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 T2 beta III, were retained by troponin C-Sepharose 4B in the presence of troponin I, even in the solution containing 1.0 M NaCl, in the presence and absence of Ca2+ ion. On the basis of these findings, the interactions among troponin components and tropomyosin are discussed.