We have previously shown that CD82, a transmembrane protein of the tetraspanin superfamily is associated with EGFR and has a negative effect on EGF-induced signalling (Odintsova, E., Sugiura, T. and Berditchevski, F. (2000) Curr. Biol. 10, 1009-1012). Here we demonstrate that CD82 specifically attenuates ligand-induced dimerization of EGFR. The recombinant soluble large extracellular loop of CD82 has no effect on the dimerization thereby suggesting that other parts of the protein are required. Although CD82 is also associated with ErbB2 and ErbB3, ligand-induced assembly of the ErbB2-ErbB3 complexes is not affected in CD82-expressing cells. Furthermore, in contrast to the CD82-EGFR association, CD82-ErbB2 and CD82-ErbB3 complexes are stable in the presence of ErbB3 ligand. The effect of CD82 on the formation of EGFR dimers correlates with changes in compartmentalisation of the ErbB receptors on the plasma membrane. Expression of CD82 causes a significant increase in the amount of EGFR and ErbB2 in the light fractions of the sucrose gradient. This correlates with the increased surface expression of gangliosides GD1a and GM1 and redistribution of GD1a and EGFR on the plasma membrane. Furthermore, in CD82-expressing cells GD1a is co-localised with EGFR and the tetraspanin. Taken together our results offer a molecular mechanism of the attenuating activity of CD82 towards EGFR, whereby GD1a functions as a mediator of CD82-dependent compartmentalisation of the receptor.