Platelet cytoskeletons were isolated by extracting these highly contractile cells with a solution containing 1% Triton X-100 and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid as recently described (Rosenberg, S., Stracher, A., and Lucas, R. C. (1981) J. Cell Biol. 91, 201-211). The Triton-insoluble cytoskeleton consists mostly of actin, a high molecular weight actin-binding protein and a previously unidentified protein with an apparent molecular weight on sodium dodecyl sulfate gels of 105,000 (+/- 5,000). We describe the purification of this 105,000-dalton protein from the platelet cytoskeleton using ammonium sulfate fractionation and ion exchange chromatography. This 105,000-dalton protein was found to cross-react with antibodies to beef cardiac alpha-actinin. One-dimensional partial proteolysis maps showed similarity to, but not identity with, the major peptides of the platelet 105,000-dalton protein and skeletal muscle alpha-actinin. The platelet 105,000-dalton cytoskeletal protein binds to and causes the sedimentation of skeletal muscle F-actin under comparatively low centrifugal force. This process, however, is inhibited by calcium ions, unlike the binding of any of the muscle alpha-actinins described to date. Thus, it is likely that the 105,000-dalton protein is the platelet form of alpha-actinin, its different structure accounting for its different actin-binding behavior.