Actin filament formation during early stages of platelet activation by stimulants was analyzed by determining the distributions of cytoskeletal proteins to a low speed centrifuge pellet (TP), ultracentrifuge pellet (UP), and ultracentrifuge supernatant (US) after lysing platelets with 1% Triton X-100. TP contained actin binding protein, myosin, alpha-actinin and actin, and UP contained these cytoskeletal proteins and other proteins. During thrombin activation, total protein and actin in TP increased with time, while they decreased in UP. Aggregating agents (ADP, PMA, thrombin) and, to a small extent, colchicine and nocodazole, microtubule inhibitors, increased cytoskeletal proteins and total protein in TP, while cytochalsin B, an inhibitor of actin polymerization, had the opposite effect. When the amounts of the respective proteins in TP and UP were summed, these values were not affected by agonists and inhibitors, except in the case of thrombin stimulation. These data suggest that the actin in UP is a form intermediate between the actin filaments in TP and actin monomer in the soluble fraction, and there may be a dynamic conversion between these forms upon platelet activation. UP was also characterized by immunostaining with antibodies against fibrinogen, tubulin, and glycoprotein Ib after SDS-PAGE/electrophoretic transfer to nitrocellulose sheets.