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Activation of protein kinase C (PKC)-A decreases normal and neoplastic cell proliferation by inhibiting epidermal growth factor receptor (EGFR)-related signaling. The molecular interactions upstream to PKC-A that influence its suppression of EGFR, however, are poorly understood. We have found that caveolin-1, tetraspanin CD82, and ganglioside GM3 enable the association of EGFR with PKC-A, ultimately leading to inhibition of EGFR signaling. GM3- and CD82-induced inhibition of EGFR signaling requires PKC-A translocation and serine/threonine phosphorylation, which eventually trig-gers EGFR Thr654 phosphorylation and receptor internaliza-tion. Within this ordered complex of signaling molecules, the ability of CD82 to associate with PKC-A requires the presence of caveolin-1, whereas the interaction of caveolin-1 or PKC-A with EGFR requires the presence of CD82 and ganglioside GM3. Disruption of the membrane with methyl-B-cyclodextrin dissociates the EGFR/GM3/caveolin-1/CD82/PKC-A complex and prevents the inhibitory effect of PKC-A on EGFR phos-phorylation, suggesting that caveolin-1, CD82, and ganglioside interact with EGFR and PKC-A within intact cholesterol-enriched membrane microdomains. Given the role of these membrane molecules in suppressing EGFR signaling, up-regulation of GM3, caveolin-1, and CD82 function may be an effective adjunctive therapy for treating epithelial cell malignancies. [Cancer Res 2007;67(20):9986–95