The earliest known step in the activation of the high affinity IgE receptor, FcepsilonRI, is the tyrosine phosphorylation of its beta and gamma subunits by the Src family tyrosine kinase, Lyn. We report here that aggregation-dependent association of FcepsilonRI with specialized regions of the plasma membrane precedes its tyrosine phosphorylation and appears necessary for this event. Tyrosine phosphorylation of beta and gamma occurs in intact cells only for FcepsilonRI that associate with these detergent-resistant membrane domains, which are enriched in active Lyn. Furthermore, efficient in vitro tyrosine phosphorylation of FcepsilonRI subunits occurs only for those associated with isolated domains. This association and in vitro phosphorylation are highly sensitive to low concentrations of detergent, suggesting that lipid-mediated interactions with Lyn are important in FcepsilonRI activation. Participation of membrane domains accounts for previously unexplained aspects of FcepsilonRI-mediated signaling and may be relevant to signaling by other multichain immune receptors.