High expression of human epidermal growth factor receptor 2 (HER2) in breast and gastroesophageal carcinomas is a predictive biomarker for treatment using HER2-targeted therapeutics (antibodies trastuzumab and pertuzumab, antibody-drug conjugate trastuzumab DM1, and tyrosine kinase inhibitor lapatinib). Radionuclide molecular imaging of HER2 expression might permit stratification of patients for HER2-targeting therapies. In this study, we evaluated a new HER2-imaging probe based on the designed ankyrin repeat protein (DARPin) 9-29. DARPin 9-29 was labeled with iodine-125 by direct radioiodination and with [99mTc]Tc(CO)3using the C-terminal hexahistidine tag. DARPin 9-29 preserved high specificity and affinity of binding to HER2-expressing cells after labeling. Uptake of [125I]I-DARPin 9-29 and [99mTc]Tc(CO)3-DARPin 9-29 in HER2-positive SKOV-3 xenografts in mice at 6 h after injection was 3.4 ± 0.7 %ID/g and 2.9 ± 0.7 %ID/g, respectively. This was significantly (p