Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a β₃-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the α subunit partner of β₃ integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that α_v forms an α_vβ₃ heterodimer in DI TNC1 astrocytes was obtained. In neuron–astrocyte association assays, the presence of either anti-α_v or anti-β₃ integrin antibodies reduced cell–cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron–astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with α_vβ₃ integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the α_vβ₃ integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.