Cardioglobulin is a protein system in mammalian plasma, assayed by its effect on contractility of isolated frog heart. Gel filtration was employed to separate individual components, called cardioglobulin-A, -B and -C, from rat and human plasma: Cardioglobulin-A of both rat and human is a small globulin. Biological activity is abolished by heparin. A part of rat plasma cardioglobulin-A probably exists as a readily dissociable complex with another protein and is not inactivated by heparin. Rat cardioglobulin-C is a large globulin, contains calcium essential for biological activity, and binds strongly to the frog heart. The human protein that corresponds functionally to rat cardioglobulin-C is a much smaller globulin and does not bind. Both rat and human cardioglobulin-B are sensitive to heparin at concentrations recommended for collection of plasma but are stable in tenfold lower concentrations that still prevent clotting. Human cardioglobulin-B separates into two fractions on G-200 Sephadex. It is not known whether they exist as a complex in plasma or as individual proteins. Rat cardioglobulin-B has not been isolated, but preliminary data indicate it is chemically distinct from human cardioglobulin-B. As a result of this work, assay of individual Cardioglobulin components can be reliably performed.