Platelet activation by adenosine diphosphate (ADP) results in changes in the composition of the large cytoskeletal fragments that can be isolated following solubilization of platelets with Triton X-100 and low speed centrifugation. Here we have used several different agents that modify platelet responses to investigate some of the factors that affect these cytoskeletal changes. All the experiments involved use of hirudinized platelet-rich plasma in which TXA, synthesis and release of dense body constituents does not occur following platelet activation with ADP. ADP alone caused a significant and sustained increase in the cytoskeletal content of actin binding protein (ABP), myosin,α-actinin, a 66K protein and actin, and a significant decrease in a 31K protein. In the presence of MK-852 or GR 144053 (GpIIbDIIa antagonists), in samples merely left unstirred and in Glanzmann's thrombasthesenia, ADP produced no increase in ABP or the 66K protein and no decrease in the 31K protein. The increase in myosin andα-actinin became reversible but there was still incorporation of actin into the cytoskeleton. In the presence of ARL 66096 (a P2Tpurinoceptor antagonist that inhibits aggregation but not shape change) there was no increase in ABP or the 66K protein and no decrease in the 31K protein. ARL 66096 also prevented incorporation ofα-actinin and actin. As with MK-852, myosin incorporation became reversible. Iloprost inhibited all the cytoskeletal changes, the effects of MgCI2were similar to those of MK-852, and acetylsalicylic acid (ASA) had no effect. In some experiments MK-852, ARL 66096, iloprost or MgCI, were added 0.5 min after the ADP. They all produced disaggregation and this was accompanied by reversal of the changes in the composition of the cytoskeleton that had occurred initially on stimulating the platelets with ADP. The results suggest that: (1) myosin is incorporated into the cytoskeleton transiently during shape change; (2) ADP interaction with the P2Treceptor leads to incorporation ofα-actinin and actin into the cytoskeleton as well as platelet aggregation; (3) further incorporation ofα-actinin and myosin and incorporation of ABP and the 66K protein occur consequent to fibrinogen binding and platelet aggregation; (4) displacement of the 31K protein from the cytoskeleton is also a consequence of fibrinogen binding and platelet aggregation; (5) platelet disaggregation is accompanied by reversal of any cytoskeletal changes that have already occurred; (6) continuous occupation of the P2Treceptor is required for maintenance of the cytoskeletal changes; (7) CAMP inhibits and reverses cytoskeletal assembly; and (8) MgCl2acts similarly to a GpIIb/IIIa antagonist under these experimental conditions.