Zirconium-89 (Zr-89) is a suitable radionuclide "for positron-emission tomography (PET) of long-circulating targeting vectors such as monoclonal antibodies (mAbs). Due to stability concerns for the most widely used 89Zr-chelating agent desferrioxamine B (DFO) in preclinical studies, alternative Zr-89-chelators are currently being developed. We recently reported on the first tetrakis(3-hydroxy-4-pyridinone) (3,4-HOPO) ligand THPN, which was identified as a promising Zr-89-chelator. In this study, we aimed to further explore this octadentate chelate in vitro and in vivo. The [Zr-IV(THPN)] thermodynamic stability was quantified in solution titration studies, which revealed one of the highest formation constants reported for a zirconium chelate (log beta(mL) 50.3(1), pM = 42.8). Solution stabilities with iron(III) were also exceptionally high and can compete with some of the strongest Felt-chelates. A first bifunctional derivative of the octadentate ligand, p-SCN-Bn-THPN, was then produced in a multistep synthesis. To assess and compare the long-term Zr-89 complex stability, bifunctional THPN, as well as the literature chelators p-SCN-Phe-DFO and p-SCN-Phe-DFO*, were conjugated to the high-molecular weight (800 kDa) polymeric carrier hyperbranched polyglycerol (HPG). The functionalized HPGs were radiolabeled with Zr-89(IV), and the integrity of the radioconjugates was assessed over several days in vitro and in vivo. While all three radioconjugates remained >95% intact over 5 days in human plasma, the in vivo study in healthy mice revealed higher physiologic stability of the DFO and DFO* radiochelates over bifunctional THPN conjugates. This was evidenced by increased bone uptake of osteophilic Zr-89(IV) for THPN. This finding contrasts with the exceptionally high thermodynamic stability of the chelate and suggests either a kinetic or metabolic lability, or may stem from coordinative changes due to the covalent conjugation of the Zr-89-THPN radiochelate as suggested by density functional theory (DFT) calculations. These important findings inform the design of next generation 3,4-HOPO chelates with the aim of improving the physiologic stability. This study furthermore demonstrates how HPG can be used as a robust carrier tool to assess and compare the long-term in vivo stability of radiochelates.