Abstract Background There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [ 89 Zr]Zr-oxine (8-hydroxyquinoline) and [ 89 Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. Procedures Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [ 89 Zr]Zr-oxine or [ 89 Zr]Zr-DFO-NCS. The cellular retention of 89 Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. Results The optimised synthesis of [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89 Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1–8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells’ proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. Conclusions Our study demonstrates that [ 89 Zr]Zr-oxine and [ 89 Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [ 89 Zr]Zr-oxine was superior to [ 89 Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.