Four fragments of alpha-tropomyosin were prepared by specific cleavage at the Cys 190 by 2-nitro-5-thiocyanobenzoic acid and by tryptic digestion. These fragments were called the N-chain corresponding to residue 1 to 189 of the original chain, the C-chain from 190 to 284, the s-fragment from 13 to 149 and/or 128, and the p-fragment from 183 to 284, respectively. Fragments individually have little binding capacity to troponin as shown by gel electrophoresis. But a new band of the complex with troponin was detected using mixtures of the fragments, one from the N-terminal side and the other from the C-terminal side, i.e., the N- and C-chains, the s- and p-fragments, the N-chain and the p-fragment, and the s-fragment and the C-chain. Therefore, the troponin binding region of tropomyosin is thought to be located between residues 150 and 190.