The threshold, extent and termination of TCR activation is controlled in part by inhibitory co-receptors expressed on activated T cells. The lymphocyte activation gene product (LAG-3), a ligand for MHC class II molecules co-caps with the CD3/TCR complex and inhibits cell proliferation and cytokine secretion in response to CD3 signaling. We first investigated whether LAG-3 is localized in activated T cells in detergent-resistant membrane rafts enriched in glycosphingolipids and cholesterol. We showed that both LAG-3 and MHC class II are present in the cell fraction of glycosphingolipid-rich complexes (GSL complexes) before the assembly of the immunological synapse by CD3/TCR complex cross-linking. Using the LAG-3 intracytoplasmic region as bait in the yeast two-hybrid cloning system, we next identified a novel protein termed LAP for LAG-3-associated protein. LAP is encoded by a 1.8-kb RNA message in lymphocytes and encodes a 45-kDa protein that is expressed in most tissues. We showed that LAP binds specifically in vitro and in vivo to the Glu-Pro (EP) repeated motif present in the LAG-3 intracytoplasmic region. LAP also binds to the EP motif of another functionally important receptor, the PDGFR. Thus, LAP is a candidate molecule for a new type of signal transduction and/or coupling of clustered rafts to the microtubule networks that could explain how negative signaling of co-receptors may occur through molecules devoid of any immunoreceptor tyrosine-based inhibitory motif consensus sequence.