Introduction:131I-GMIB-Anti-HER2-VHH1 is a targeted radionuclide theranostic agent directed at HER2 expressing cancers. VHH1 is a single domain antibody fragment covalently linked to therapeutic radio-iodine 131I via the linker SGMIB. The Phase I study presented was aimed at evaluating the safety, biodistribution, radiation dosimetry and tumor imaging potential of 131I-GMIB-Anti-HER2-VHH1 in healthy volunteers and breast cancer patients. Methods: In a first cohort, six healthy volunteers were included. The biodistribution of 131I-GMIB-Anti-HER2-VHH1 was assessed using whole body (anterior and posterior) planar images obtained at 40 min., 2, 4, 24 and 72 h following i.v. administered (38 ± 9 MBq) 131I-GMIB-VHH1. Imaging data were analyzed using OLINDA/EXM software 1.0 to determine the dosimetry. Blood and urine samples were obtained over 72h. In the second cohort, three patients with metastatic HER2 positive breast cancer were included. Planar whole-body imaging was performed at 2 h and 24 h after injection. Additional SPECT/CT images were obtained following the whole body images at 2 and 24 h in case of relevant uptake in known cancer lesions Results: No drug related adverse events (AEs) were observed throughout the study. The biological half-life of 131I-GMIB-Anti-HER2-VHH1 in healthy subjects was about 8 h. After i.v. administration, the compound is eliminated from the blood with a 2.5 h half-life. The drug is primarily eliminated via the kidneys. The drug was stable in circulation and there was no increased accumulation in thyroid or stomach. The absorbed dose to the kidneys was 1.54 ± 0.25 mGy/MBq, while to bone marrow 0.03 ± 0.01 mGy/MBq. SPECT/CT imaging in patients with advanced breast cancer showed focal uptake of 131I-GMIB-Anti-HER2-VHH1 in metastatic lesions. Conclusion: No AEs were observed after iv administration of 131I-GMIB-Anti-HER2-VHH1 at low activity. Unbound drug is rapidly eliminated via the kidneys. In patients with stage IV HER2 positive breast cancer accumulation of 131I-GMIB-Anti-HER2-VHH1 in metastatic sites was observed. Dosimetry predicts kidneys as the dose limiting organ upon dose escalation, but kidney toxicity should only occur at very high injected activities. Dose escalation is planned in a subsequent phase I/II study to assess the therapeutic window of this compound (NCT04467515).