We have investigated raft formation in human platelets in response to cell activation. Lipid phase separation and domain formation were detected using the fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (diI-C18) that preferentially partitions into gel-like lipid domains. We showed that when human platelets are activated by cold and physiological agonists, rafts coalesce into visible aggregates. These events were disrupted by depletion of membrane cholesterol. Using Fourier transform infrared spectroscopy (FTIR), we measured a thermal phase transition at around 30°C in intact platelets, which we have assigned as the liquid-ordered to the liquid-disordered phase transition of rafts. Phase separation of the phospholipid and the sphingomyelin-enriched rafts could be observed as two phase transitions at around 15 and 30°C, respectively. The higher transition, assigned to the rafts, was greatly enhanced with removal of membrane cholesterol. Detergent-resistant membranes (DRMs) were enriched in cholesterol (50%) and sphingomyelin (20%). The multi-functional platelet receptor CD36 selectively partitioned into DRMs, whereas the GPI-linked protein CD55 and the major platelet integrin αIIbβ3a did not, which suggests that the clustering of proteins within rafts is a regulated process dependent on specific lipid protein interactions. We suggest that raft aggregation is a dynamic, reversible physiological event triggered by cell activation. J. Cell. Physiol. 190: 117–128, 2002. © 2002 Wiley-Liss, Inc.