Human and rabbit platelets fully aggregated by platelet-activating factor (PAF) underwent slow disaggregation but were rapidly disaggregated by the PAF receptor antagonists WEB-2086, Y-24180, SM-12502, and CV-3988. Whereas the 1-alkyl-2-[³H]acetyl-sn-glycero-3-phosphocholine ([³H]acetyl-PAF) specifically bound to platelet receptors underwent slow and spontaneous dissociation, it dissociated promptly from its receptor when WEB-2086 was added, in parallel with platelet disaggregation and disappearance of P-selectin on the cell surface. Extracellular [³H]acetyl-PAF was rapidly deacetylated by normal rabbit platelets; some of the [³H]acetyl-PAF was bound to the cells and a very small amount of [³H]acetate was detected in the cells. In contrast, when 1-[³H]alkyl-2-acetyl-sn-glycero-3-phosphocholine was added to the platelets, the radioactivity was rapidly incorporated into the 1-alkyl-2-acyl-sn-glycero-3-phosphocholine fraction. These results indicate that 1) continuous binding of PAF to its receptor is necessary for prolonged platelet aggregation, which may be mediated through an unknown signaling system for a long-term cell response rather than a transient signaling system, and 2) most of the [³H]acetyl-PAF bound to platelets is metabolized extracellularly by ecto-type PAF acetylhydrolase, with the lyso-PAF generated being incorporated rapidly into the cells and converted to 1-alkyl-2-acyl-sn-glycero-3-phosphocholine.