BACKGROUND Pretargeted radioimmunoimaging and -therapy approaches building on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have yielded impressive results in recent years and have proven a vital alternative to biological pretargeting systems. After improvement of the TCO-antibody conjugates, we here report on our evaluation of a new series of radiolabeled Tz-probes. METHODS Four new Tz-probes were synthesized, radiolabeled with lutetium-177, and characterized in vitro in terms of lipophilicity, reactivity, and stability in PBS and mouse serum. The in vivo biodistribution profile and tumor-targeting potential of the probes were evaluated in LS174T tumor-bearing mice pretargeted with TCO-antibody conjugates using non-pretargeted mice as control. RESULTS Radiolabeling of all probes proceeded in high yields providing the 177Lu-labeled tetrazines in >95% radiochemical purity without any further purification. In mouse serum, half-lives of the probes varied between 8 and 13 h, with the exception of the most lipophilic probe, [177Lu]1b, with a serum half-life of less than 1 h. This probe also showed the fastest blood clearance (t1/2 = 5.4 min), more than 2-fold faster than PEG-linked probes [177Lu]3 and [177Lu]4, and even 3-fold faster than the other small probes without the PEG-linker, [177Lu]1a and [177Lu]2. In the pretargeting experiments, tumor uptake of the lead probe [177Lu]4 (~6 %ID/g) was most closely approached by [177Lu]2, followed by [177Lu]3 and [177Lu]1a. While all the smaller and more lipophilic probes suffered from increased liver uptake, the PEG-linked probe [177Lu]3 with its additional negative charge surprisingly showed the highest kidney uptake among all of the probes. CONCLUSIONS The in vitro performance of some of the new tetrazine probes turned out to be comparable to the established lead probe [177Lu]Lu-DOTA-PEG11-Tz ([177Lu]4). However, tumor pretargeting studies in vivo showed lower tumor uptake and increased uptake in non-target organs.