Fragments derived from rabbit skeletal troponin T (Tn-T) were tested for binding on a troponin C (Tn-C)-Sepharose affinity column in order to locate the binding site of Tn-C on Tn-T. The COOH-terminal fragments P2 (residues 159-209) and B2 (residues 206-258) bound most strongly, confirming the earlier proposal that the highly basic COOH-terminal region of Tn-T may serve as a site of interaction for the acidic Tn-C protein. Results from circular dichroism experiments on the large fragments B1 (residues 1-205), B2, P1 (residues 91-154) and P2 indicated that most of the .alpha.-helical structure resides in CB2 (residues 71-151), the tropomyosin (Tm) binding site of Tn-T, while the remainder of the molecule including the Tn-C binding region (approximately residues 159-259) contains very little in the way of .alpha. and .beta. structure. The presence of Ca resulted in a stronger interaction between these fragments and Tn-C, illustrating the Ca-sensitive nature of this system. The addition of Mg2+ to the buffer system did not affect this binding.