The binding site for rabbit skeletal alpha-tropomyosin on troponin-T has been localized to the cyanogen bromide fragment, CB2 (residues 71-151), using affinity chromatography. The entire fragment is required to bring about the correct secondary structure conducive to binding. CB2 contains about 80% alpha-helix and accounts for most of the helical content found in troponin-T (35%). The molecular weight of CB2 obtained by sedimentation equilibrium (9700) agrees closely with the value calculated from sequence analysis (9850). Circular dichroism and sedimentation velocity experiments indicate that the helix is stable and not affected by salt concentrations of 0.1 to 1.6 M KCl nor by composition of the buffer. The helical content is unaffected by pH from 3.3 to 9.1 but decreases at pH 10-11. Temperature denaturation studies CB2 and troponin-T show that both are similarly heat labile, with loss of 50% of the ellipticity at 39 degrees C. Binding of CB2 to alpha-tropomyosin occurs in the pH range of 5.0 to 9.1, but not at pH 3-4 or 10-11. It is concluded that the helical region of CB2, and perhaps the carboxyl side chains of aspartic and glutamic acids, may be involved in binding over a limited surface area of the double-stranded coiled coil of alpha-tropomyosin.