C-proteins have been isolated from rabbit red skeletal muscle (soleus and semitendinosus) and cardiac muscle and their structure and properties compared with those of white muscle C-protein. The Mr of white, red, and cardiac C-proteins are 135,000, 145,000, and 150,000, respectively, and their s20,w values are 4.3, 3.8, and 4.8 S, indicating that red C-protein is more asymmetric than the other two. They elute quite differently from hydroxylapatite columns. Two-dimensional CNBr peptide maps show extensive differences in primary structure, and anti-white C-protein does not precipitate red or cardiac C-protein. Despite these structural differences, all three C-proteins bind equally to white, red, or cardiac myosin and to actin. All three have the same effects on actomyosin ATPase in 50 mM KCl; they inhibit red and white skeletal actomyosins but slightly activate cardiac actomyosin. X-protein, a 140,000-dalton contaminant of white C-protein, was also investigated. It is very similar to red C-protein in elution from hydroxylapatite columns, S20,w, amino acid composition, and primary structure, but small differences in Mr and peptide maps indicate that the two proteins are probably not identical.