Previous studies have demonstrated that rabbit skeletal tropomyosin consists of two or more chemically non-identical but highly homologous polypeptide chains. Attempts by a variety of techniques to prepare pure tropomyosin chains in amounts adequate for chemical characterization have been unsuccessful to date. To provide more extensive information for the purpose of elucidating the relationship between amino acid sequence and the coiled-coil structure of tropomyosin, a cyanogen bromide treatment of the S-carboxymethylated protein was carried out. The fragments were separated into small and large components by gel filtration on Sephadex G-50. The small fragments were fractionated by ion-exchange chromatography and electrophoresis on paper and their sequences elucidated by conventional methods. Coupled with previous data, these results indicate a minimum of seven unique methionine sequences and are consistent with a high degree of homology in the tropomyosin polypeptide chains. From the mixture of the larger cyanogen bromide polypeptides, a fragment was isolated by ion-exchange chromatography on QAE-Sephadex. In aqueous buffer it had a molecular weight of 35 000 and an α-helical content of about 60% as estimated by circular dichroism. In 8 M urea its molecular weight was reduced to 15 000, a value in reasonable agreement with a minimal molecular weight of 17 000 calculated from its amino acid composition. From its histidine content (two residues) and the known COOH-terminal amino acid sequence of the protein, the fragment was concluded to be derived from the COOH-terminal half of the molecule. These results are consistent with a degree of 'coiled-coil' structure in a fragment representing about one-half of the tropomyosin molecule.