Affibody® ligands to the extracellular domain of human epidermal growth factor receptor 2 (HER2), which is known to be overexpressed in ~ 20-30% of breast cancers, have been isolated by phage display in vitro selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants selectively bound with nanomolar affinity (KD ~50 nM) to the extracellular domain of HER2. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, that was shown to have an improved apparent affinity to HER2 (KD~ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled affibody ligands showed specific binding in vitro to native HER2 molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors in vivo, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as in vivo imaging and radiotherapy.