BACKGROUND:  Bladder cancer remains a significant global health challenge, with approximately 75% of cases presenting as non-muscle-invasive bladder cancer. Despite standard treatment with transurethral resection and intravesical Bacillus Calmette-Guérin immunotherapy, up to 40% of patients develop resistance or progress to muscle-invasive disease. Targeted alpha-emitting radionuclide therapy offers promising therapeutic potential through the selective delivery of high linear energy transfer radiation to tumor cells while minimizing damage to healthy tissues. PTK7 is overexpressed in various malignancies, including bladder cancer, and is therefore a viable therapeutic target. This study evaluated the preclinical efficacy of [(212)Pb]Pb-TCMC-chOI-1, a (212)Pb-labeled antibody targeting PTK7, for targeted alpha-emitting radionuclide therapy in bladder cancer using 2D adherent cultures (clonogenic assay) and 3D multicellular spheroid models (spheroid growth inhibition). RESULTS:  PTK7 expression analysis revealed varying antigen densities across five bladder cancer cell lines, ranging from approximately 10,000 to 70,000 sites per cell. The chimeric anti-PTK7 antibody demonstrated apparent equilibrium dissociation constants of 10-44 nM with moderate binding affinity suitable for therapeutic applications. [(212)Pb]Pb-TCMC-chOI-1 treatment resulted in activity- and time-dependent cytotoxicity, with enhanced sensitivity observed in cell lines with higher PTK7 levels. In clonogenic assays, the activity concentration required for 50% growth reduction was 48-74 kBq/mL, corresponding to 22-51 bound and 9-16 internalized (212)Pb atoms per cell. In 3D models, similar therapeutic effects were observed despite significantly lower activities (values of approximately 1 and 30 kBq/mL for KU-19-19 and 647-V cells, respectively), suggesting a more pronounced cross-fire effect. Flow cytometry demonstrated treatment-induced DNA damage, cell cycle perturbations and cell death, with response patterns correlating with overall treatment sensitivity. RT-112 and KU-19-19 cells showed superior responses compared to 647-V and T-24 cells, consistent with their higher PTK7 expression. CONCLUSIONS:  These findings support PTK7 as a therapeutic target for bladder cancer and demonstrate the potential of [(212)Pb]Pb-TCMC-chOI-1 for targeted alpha-emitting radionuclide therapy. The results provide a rationale for further preclinical optimization of this therapeutic approach. Trial registration number (TRN): Not applicable.