Adding adenosine diphosphate (ADP) to platelet-rich plasma (PRP) results in a fall in the level of platelet monomeric globular (G)-actin indicative of actin polymerization. There is an immediate fall in G-actin associated with shape change which is reversible, and a second phase or sustained response associated with second phase or irreversible aggregation. Previous studies suggested that platelet aggregation is a prerequisite for second phase or sustained actin polymerization. Here we have examined further the relationship between platelet aggregation and actin polymerization in ADP-stimulated platelets by studying the effects of M148, a monoclonal antibody that inhibits aggregation by combining with the glycoprotein (Gp) IIb/IIIa complex, and the effects of dissociating GpIIb/IIIa by incubating platelets with EGTA at 37°C. We also assessed the contribution of thromboxane A(2) (TXA(2)) by inhibiting its synthesis with aspirin. The results show that GpIIb/IIIa is involved in mediating the second phase or sustained actin polymerization that occurs after activating platelets with ADP and confirm the requirement for platelet aggregation. TXA(2) synthesis is not required for second phase or sustained actin polymerization, but TXA(2) contributes to second phase or sustained actin polymerization, probably via promotion of further platelet-platelet contact.