CD44 is involved in several biological processes owing to its dual role as a cell adhesion and signaling molecule. In an allogeneic dendritic cell (DC)-T cell interaction model, we show here that CD44 gets clustered at the contact between T cells with mature but not immature DCs. Also, CD44 colocalized with lipid rafts at the immunological synapse (IS). Using DCs or T cells derived from CD44-deficient mice, we observed that the presence of CD44 on DCs and T cells is important for the formation of DC-T cell tight conjugates. However, deficiency of CD44 on DCs but not T cells affected the functional IS, as indicated by decreased phosphotyrosine and protein kinase C-theta enrichment at the synapse. Also, CD44-deficient DCs induced significantly decreased proliferation as well as IL-2 and IFN-gamma production from allogeneic T cells. The polarization of CD44 at the synapse was also noted in an antigen (OVA)-specific, syngeneic DC-T cell interaction using OVA-specific T cells derived from OT-II mice. It was believed that large molecules such as CD44 were excluded from the IS. Results presented here show for the first time that CD44 is recruited to the IS during allogeneic DC and T cell interactions and plays an important role in subsequent T cell activation.