The activation of the epidermal growth factor receptor (EGFR) kinase requires ligand binding to the extracellular domain (ECD). Previous reports demonstrate that the EGFR-ECD can be crystallized in two conformations - a tethered monomer or, in the presence of ligand, an untethered back-to-back dimer. We use Biosensor analysis to demonstrate that even in the monomeric state different C-terminal extensions of both truncated (EGFR(1-501))-ECD and full-length EGFR(1-621)-ECD can change the conformation of the ligand-binding site. The binding of a monoclonal antibody mAb806, which recognizes the dimer interface, to the truncated EGFR(1-501)-Fc fusion protein is reduced in the presence of ligand, consistent with a change in conformation. On the cell surface, the presence of erythroblastosis B2 (erbB2) increases the binding of mAb806 to the EGFR. The conformation of the erbB2: EGFR heterodimer interface changes when the cells are treated with epidermal growth factor (EGF). We propose that ligand induces kinase-inactive, pre-formed EGFR dimers and heterodimers to change conformation leading to kinase-active tetramers, where kinase activation occurs via an asymmetric interaction between EGFR dimers.