The human epidermal growth factor receptor 3 (HER3) is an interesting target for antitumor therapy. For optimal HER3 signaling inhibition, a biparatopic Nanobody construct (MSB0010853) was developed that binds 2 different HER3 epitopes. In addition, MSB0010853 contains a third HER3 epitope that binds albumin to extend its circulation time. MSB0010853 is cross-reactive with HER3 and albumin of mouse origin. We aimed to gain insight into MSB0010853 biodistribution and tumor uptake by radiolabeling the Nanobody construct with Zr-89. Methods: MSB0010853 was radiolabeled with Zr-89. Dose-and time-dependent tumor uptake was studied in nude BALB/c mice bearing a subcutaneous HER3 overexpressing H441 non-small cell lung cancer xenograft. Dose-dependent biodistribution of Zr-89-MSB0010853 was assessed ex vivo at 24 h after intravenous injection. Protein doses of 5, 10, 25, 100, and 1,000 mgwere used. Time-dependent biodistribution of MSB0010853 was analyzed ex vivo at 3, 6, 24, and 96 h after intravenous administration of 25 mg of Zr-89-MSB0010853. PET imaging and biodistribution were performed 24 h after administration of 25 mu g of Zr-89-MSB0010853 to mice bearing human H441, FaDu (high HER3 expression), or Calu-1 (no HER3 expression) tumor xenografts. Results: Radiolabeling of MSB0010853 with Zr-89 was performed with a radiochemical purity of greater than 95%. Ex vivo biodistribution showed protein dose-and time-dependent distribution of Zr-89-MSB0010853 in all organs. Uptake of Zr-89-MSB0010853 in H441 tumors was only time-dependent. Tumor could be visualized up to at least 96 h after injection. The highest mean SUV of 0.6 +/- 0.2 was observed at 24 h after injection of 25 mu g of Zr-89-MSB0010853. Zr-89-MSB0010853 tumor uptake correlated with HER3 expression and was highest in H441 (6.2 +/- 1.1 percentage injected dose per gram [% ID/g]) and lowest in Calu-1 (2.3 +/- 0.3 % ID/g) xenografts. Conclusion: Zr-89-MSB0010853 organ distribution and tumor uptake in mice are time-dependent, and tumor uptake correlates with HER3 expression. In contrast to tumor uptake except for kidney uptake, organ distribution of Zr-89-MSB0010853 is protein dose-dependent for the tested doses. Zr-89-MSB0010853 PET imaging gives insight into the in vivo behavior of MSB0010853.