We have isolated a tropomyosin binding protein (TMBP) from human platelets using isoelectric fractionation, hydroxylapatite chromatography, and affinity chromatography on skeletal muscle tropomyosin-Affi-Gel 15. TMBP is a 67,000-Da monomeric protein that binds to muscle and nonmuscle tropomyosin affinity resins. Its affinity for platelet tropomyosin is greater than for rabbit skeletal or chicken gizzard tropomyosin, and greater than that of troponin for all tropomyosin affinity resins tested. TMBP forms a complex with platelet tropomyosin that can be isolated on G-150. The approximate molar stoichiometry is 1:1. Troponin and TMBP have distinct binding sites on skeletal tropomyosin since binding of TMBP to tropomyosin-Affi-Gel 15 is not affected by previous saturation of the column with troponin (or vice versa). The amino acid composition of TMBP is virtually identical with that of human serum albumin, and is similar to those of beta-actinin (Heizmann, C. W., Müller, G., Jenny, E., Wilson, K. J., Landon, F., and Olomucki, A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 74-77) and acumentin (Southwick, F. S., and Stossel, T. P. (1981) J. Biol. Chem. 256, 3030-3036). The protein we have isolated is the first nonmuscle protein other than actin that has been shown to bind to tropomyosin. Results in an accompanying paper show that this tropomyosin binding protein is identical with human serum albumin (Hitchcock-DeGregori, S. E., Gerhard, M. D., and Brown, W. E. (1985) J. Biol. Chem. 260, 3228-3231).