Thy-1 (CD90) is a glycoprotein bound to the plasma membrane by a GPI anchor. Aggregation of Thy-1 in mast cells and basophils induces activation events independent of the expression of Fcepsilon receptor I (FcepsilonRI). Although we and others have previously suggested that plasma membrane microdomains called lipid rafts are implicated in both Thy-1 and FcepsilonRI signaling, properties of these microdomains are still poorly understood. In this study we used rat basophilic leukemia cells and their transfectants expressing both endogenous Thy-1.1 and exogenous Thy-1.2 genes and analyzed topography of the Thy-1 isoforms and Thy-1-induced signaling events. Light microscopy showed that both Thy-1 isoforms were in the plasma membrane distributed randomly and independently. Electron microscopy on isolated membrane sheets and fluorescence resonance energy transfer analysis indicated cross-talk between Thy-1 isoforms and between Thy-1 and FcepsilonRI. This cross-talk was dependent on actin filaments. Thy-1 aggregates colocalized with two transmembrane adaptor proteins, non-T cell activation linker (NTAL) and linker for activation of T cells (LAT), which had been shown to inhabit different membrane microdomains. Thy-1 aggregation led to tyrosine phosphorylation of these two adaptors. The combined data indicate that aggregated GPI-anchored proteins can attract different membrane proteins in different clusters and thus can trigger different signaling pathways.