Rabbit platelet-activating factor (PAF) is a lipid released from IgE-sensitized basophils and mast cells during challenge with antigen. PAF induces shape change, aggregation, and secretion of granule-associated 3[H]-serotonin in rabbit platelets. This study investigated the capacity of PAF to initiate rabbit platelet prostaglandin synthesis and the requirement for generated prostaglandin endoperoxides and thromboxane A2 in PAF-induced platelet aggregation and secretion. PAF in submaximal concentrations induced synthesis in rabbit platelets of PGE2, PGF2α, and prostaglandin endoperoxides in amounts comparable to that initiated by collagen, but greater than that caused by thrombin. PAF stimulation of platelets preloaded with 14[C]-arachidonic acid caused platelet production of 14[C]-thromboxane B2, the stable derivative of 14[C]-thromboxane A2. When compared kinetically to secretion, PAF induced platelet endoperoxide production peaked just before maximal release of granular 3[H]-serotonin content. However, the cyclooxygenase inhibitors, aspirin and indomethacin, failed to alter PAF-induced platelet shape change or aggregation. Similarly, secretion was unaffected by indomethacin except for a slight attenuation seen at the lowest stimulus concentrations examined. Platelet production of endoperoxides occurred throughout the range of PAF concentrations examined, even those causing barely detectable levels of secretion. PAF thus initiates platelet synthesis of prostaglandin endoperoxides and thromboxane A2, but aggregation and secretion induced by PAF occur independent of these products of the cyclooxygenase pathway.