The Affibody molecule Z(HER2:342-pep2), site-specifically and homogeneously conjugated with a 1,4,7,10-tetra-azacylododecane-N,N',N'',N'''-tetraacetic acid (DOTA) chelator, was produced in a single chemical process by peptide synthesis. DOTA-Z(HER2:342-pep2) folds spontaneously and binds HER2 with 65 pmol/L affinity. Efficient radiolabeling with >95% incorporation of (111)In was achieved within 30 min at low (room temperature) and high temperatures (up to 90 degrees C). Tumor uptake of (111)In-DOTA-Z(HER2:342-pep2) was specific for HER2-positive xenografts. A high tumor uptake of 23% injected activity per gram tissue, a tumor-to-blood ratio of >7.5, and high-contrast gamma camera images were obtained already 1 h after injection. Pretreatment with Herceptin did not interfere with tumor targeting, whereas degradation of HER2 using the heat shock protein 90 inhibitor 17-allylamino-geldanamycin before administration of (111)In-DOTA-Z(HER2:342-pep2) obliterated the tumor image. The present results show that radiolabeled synthetic DOTA-Z(HER2:342-pep2) has the potential to become a clinically useful radiopharmaceutical for in vivo molecular imaging of HER2-expressing carcinomas.