Background <p>Radioconjugates targeting the fibroblast activation protein (FAP) have been successfully applied for nuclear imaging and are currently under investigation for radionuclide therapy. The aim of this study was to assess the biophysical properties of the ¹⁷⁷Lu-labeled FAP inhibitors (FAPIs), FAPI-46, OncoFAP, FAP-2286 and 3BP-3940, and compare them with their ¹⁶¹Tb-labeled analogues. The respective ¹⁷⁷Lu- and ¹⁶¹Tb-labeled FAPIs were evaluated using fibrosarcoma tumor cells in vitro and a xenograft mouse model to demonstrate pharmacokinetic equivalence.</p> Results <p>The radioconjugates (500 MBq/mL) remained ≥ 96% intact in saline over 24&#xa0;h, irrespective of the employed radionuclide. The ¹⁶¹Tb-labeled conjugates showed similar logD values, uptake in HT1080-FAP cells and FAP-binding affinities (K_D: 0.48–1.8 nM) as their ¹⁷⁷Lu-based analogues. The in vitro binding to mouse and human blood plasma proteins was moderate with protein-bound fractions in the ranges of 39–56% and 31–61%, respectively, irrespective of the employed radionuclide. After 24 h incubation, the intact fraction of radioconjugates in mouse blood plasma was ≥ 87% while this was even ≥ 91% in human blood plasma without any differences among the same FAPIs labeled with either radionuclide. In HT1080-FAP/HT1080 tumor-bearing mice, the ¹⁶¹Tb-based FAP-targeting radioconjugates showed distribution profiles that corresponded well with their ¹⁷⁷Lu-labeled analogues as demonstrated by dual-isotope SPECT/CT imaging and quantitative biodistribution data obtained at 4&#xa0;h post-injection (p.i.). The radioconjugates were rapidly cleared from the blood, with &lt; 1.5% IA/g retained at 1&#xa0;h after administration. HT1080-FAP tumor accumulation reached its maximum with 8.8–20% IA/g at 1&#xa0;h after injection of the radioconjugates, followed by gradual wash-out to 7.4–11% IA/g at 4&#xa0;h p.i. and further to &lt; 3% IA/g at 24&#xa0;h p.i. An exception was the 3BP-3940 radioconjugate which still showed 6.4 ± 0.6% IA/g in the tumor xenograft at 24&#xa0;h p.i..</p> Conclusion <p>The in vitro and in vivo properties of the ¹⁶¹Tb-labeled radioconjugates were similar to those of the ¹⁷⁷Lu-labeled analogues. The accumulation of these FAP-targeting radioconjugates in HT1080-FAP tumors of mice was reasonably high, however, as the activity declined rapidly over time, repeated radioconjugate administration may be necessary to achieve therapeutic efficacy.</p> Graphical abstract <p></p>