The middle T-antigen (MT) encoded by polyomavirus induces tumours by mimicking an activated cell surface receptor. A hydrophobic region close to the C-terminus locates MT to the plasma membrane where it initiates oncogenic signalling. HA or FLAG tags added to the C-terminus of MT could be detected on the outside of cells, demonstrating that MT is a transmembrane protein. Addition of a KDEL sequence retained MT in the endoplasmic reticulum, where it failed to transform cells as a consequence of its lack of binding to ShcA, PI3K, and PLC-γ1 despite the phosphorylation of the appropriate binding sites. Additional studies show that MT binding to PP2A is probably required in addition to the TMD for MT to exit the ER and migrate to the plasma membrane. MT on the cell surface is present in discrete clusters that contain phosphorylated ShcA and so represent active signalling complexes. New mutations in the hydrophobic region prevented MT clustering, and also inhibited cell transformation without altering the association of MT with its known binding proteins. Overall, these data, together with previous publications, illustrate that MT associates with signalling proteins at discrete subcellular membrane sites in its maturation pathway. MT binds to PP2A in the cytoplasm, to c-Src at the ER, and to ShcA, PI3K, and PLC-γ1 at subsequent locations en route to the plasma membrane. Formation of a large macromolecular complex in the plasma membrane is probably required for MT signalling. Similar cell surface complexes are observed with activated growth factor receptors, so it is possible that normal and oncogenic signalling from receptors is also dependent upon the assembly of large macromolecular complexes at the cell surface membrane. There was no obvious association of MT clusters with lipid rafts but there was colocalisation with the actin cytoskeleton.