Rabbit skeletal tropomyosin was separated into two components, alpha and beta, by CM cellulose column chromatography in the presence of urea. The two components are apparently different from TN-T, since, 1) upon addition of the components to F-actin solutions, they increase the degree of flow birefringence delta n, while TN-T does not, 2) the reduced mean residue elipticities [theta] at 220 nm are about 2.5-fold higher than for TN-T, and they contain no proline. These features are similar to those of intact tropomyosin, but the two components are not identical for the following reasons; 1) leucine is the C-terminus of the beta component and isoleucine is the C-terminus of the alpha component, 2) the beta component has a lower helicity and a somewhate lower capacity to increase delta n of F-actin solutions than the alpha component, and 3) the beta component has a higher content of glutamic acid and methionine than the alpha component. The two components can be crystallized into paracrystals in the presence of magnesium. Electron micrographs of the paracrystals of both components show a band pattern with 400 A periodicity. Bovine cardiac tropomyosin migrates on SDS gels as two poorly resolved bands, which could be separated by CM cellulose column chromatography. The C-terminus of the slower moving component was leucine, and that of the faster moving component was isoleucine, corresponding to the beta and alpha components of skeletal tropomyosin.