We have developed a genetic method to determine the active orientation of dimeric transmembrane protein helices. The bovine papillomavirus E5 protein, a 44-amino acid homodimeric protein that appears to traverse membranes as a left-handed coiled-coil, transforms fibroblasts by binding and activating the platelet-derived growth factor (PDGF) β receptor. A heterologous dimerization domain was used to force E5 monomers to adopt all seven possible symmetric coiled-coil registries relative to one another within the dimer. Focus formation assays demonstrated that dimerization of the E5 protein is required for transformation and identified a single preferred orientation of the monomers. The essential glutamine residue at position 17 resided in the dimer interface in this active orientation. The active chimera formed complexes with the PDGF β receptor and induced receptor tyrosine phosphorylation. We also identified E5-like structures that underwent non-productive interactions with the receptor.