CD36 is a transmembrane glycoprotein receptor that engages in signal transduction implicated in important physiological and pathophysiological events. CD36 in platelets has been shown physically and functionally to associate with members of the Src family of protein tyrosine kinases, Fyn, Lyn, and Yes, but the nature of this important association has never been rigorously examined. Here, we show that CD36 does not associate with Lyn through a protein-mediated interaction. In COS cells transfected with both CD36 and Lyn these molecules did not co-precipitate, suggesting a requirement for an intermediary molecule absent from the COS cells. Yeast two-hybrid analysis confirmed that the carboxylterminal cytoplasmic tail of CD36 did not bind Lyn directly, and no Lyn binding protein bound to CD36 in a cDNA library screen. Conversely, when the CD36-Lyn association seen in platelets was analysed by biophysical parameters, dissociation occurred at 37 degrees C and also by solubilisation in octylglucoside, indicative of a lipid-mediated association. Since both CD36 and Lyn are enriched in Triton X-100-insoluble rafts at the plasma membrane, these findings point to the importance of raft-associated lipids in CD36-mediated signal transduction. (c) 2006 Elsevier Inc. All rights reserved.