Horse platelet membranes isolated by the glycerol lysis technique and subjected to SDS-PAGE showed large amounts of actin and variable amounts of myosin relative to other membrane proteins and glycoproteins. [14C]-2-dinitrothioadenosine diphosphate, when briefly incubated with whole cells, rapidly labeled the membrane actin component. Retention of myosin by the membranes during their isolation was optimized by lysing the cells and resuspending the membranes in Tris-HC1, pH 7.35, with 0.13 M KCl, 0.01 M NaCl, 2 mM MgCl2 and 0.01 mM CaCl2. Subsequently, significant amounts of actin and myosin could be eluted from the membranes with 10-3 M ADP but not with CDP, GDP or UDP. Actin was also eluted effectively from membranes prepared in Tris-NaCl, pH 7.35, by washing with 0.1 mM EDTA (in presence or absence of ADP). Despite repeated washings with either elution system, more than 50% of the actin remained associated with the membranes. When membrane vesicles with right side out (RO) and inside out (IO) orientation, separated by chromatography on Con A-Sepharose, were similarly washed identical results were obtained. Two dimensional electrophoresis of the membrane protein of IO vesicles separated two major actin components, one of which was differentially removed by prior treatment of the vesicles with 10-3 M ADP. It is concluded that (1) platelet myosin and two forms of platelet actin are associated with the cell membrane and that myosin and one form of actin can be displaced by ADP or EDTA while the second form of actin is more firmly attached, and (2) some actin is present on both membrane surfaces.