Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other proteins such as beta 1 integrins and MHC antigens. Although their functions are not clear, they have been suggested to play a role in cell adhesion and migration, signal transduction, and costimulation. We have in this paper directly compared the functional properties of four tetraspans, CD9, CD53, CD81, and CD82. mAbs to any of these molecules were able to deliver a costimulatory signal for CD3-mediated activation of the T cell line Jurkat. CD82 mAbs were the most efficient in triggering this effect. Moreover, engagement of CD9, CD81, and CD82 induced the homotypic aggregation of the megakaryocytic cell line HEL, and inhibited the migration of this cell line. Similar results were obtained with the preB cell line NALM-6 using the CD9 and CD81 mAbs. The CD81 mAb 5A6 produced the strongest effects. Therefore, the tetraspans are recognized by mAbs which produce similar effects on the same cell lines. This is consistent with the tetraspans being included in large molecular complexes and possibly forming a tetraspan network (the tetraspan web). We also demonstrate that the tetraspans are likely to keep specific functional properties inside this network. Indeed, we have demonstrated that the human CD9 is able, like the monkey molecule, to upregulate the activity of the transmembrane precursor of heparin-binding EGF as a receptor for the diphtheria toxin when cotransfected in murine LM cells. Neither CD81, nor CD82 had such activity. By using chimeric CD9/CD81 molecules we demonstrate that this activity requires the second half of CD9, which contains the large extracellular loop, the fourth transmembrane region, and the last short cytoplasmic domain.