A major question in immunology is how DC can display limited amounts of individual peptide-MHC complexes and still induce cross-linking of T-cell receptors to initiate cellular responses. One suggested mechanism is that MHC exists at the cell surface in high avidity multimers, and tetraspanin proteins, known to laterally associate with both MHC classes I and II, promote MHC multimerisation. To validate this theory, we tested the ability of DC deficient in either one of two typical tetraspanin molecules: CD37 or CD151 to present peptide to Ag-specific T cells. Surprisingly, although they exhibited no developmental or maturation defects, DC lacking either CD37 or CD151 expression were hyper-stimulatory to T cells. We demonstrate that CD37 and CD151 control DC-mediated T-cell activation by two different mechanisms: CD151 regulates co-stimulation whereas CD37 regulates peptide/MHC presentation. The implications of these results on the model suggesting that tetraspanins promote MHC multimerisation are discussed.