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Background/Objectives: The F3 peptide, a tumor-homing peptide known to bind cell-surface nucleolin, is frequently employed as a targeting vector in cancer research. However, the impact of the modification site on its cellular binding properties has not been investigated yet. In this work, we aimed to design an improved F3-based radioconjugate by identifying the optimal conjugation site and establishing a protocol for its biological evaluation in vitro. To achieve this, we compared F3 peptide derivatives modified at their N- or C-termini with DOTA for complexation of indium-111 (111In) for SPECT or Auger electron therapy or a fluorophore (FITC) for optical imaging. Methods: N-and C-terminal DOTA-modified F3 peptides were radiolabeled with indium-111 and compared for their in vitro stability in different physiologically relevant media. Suitable nucleolin-positive cell lines for further in vitro studies were identified by confocal microscopy of a FITC-labeled F3 peptide derivative. The radioconjugates were then investigated on MDA-MB-231 (breast cancer) and PC-3 (prostate cancer) cells for nucleolin-specific cell binding and uptake, and several parameters of the in vitro assays were varied to establish a suitable protocol. Results: In general, in vitro assays with F3 peptide conjugates are challenging, as the outcome depends on a number of experimental parameters, leading, in some cases, to varying results. In particular, the presence of Ca2+ and Mg2+ had a decisive impact on the results, likely because the metal ions compete with the binding of F3 conjugates to nucleolin. The C-terminal modified, 111In-labeled F3 radioconjugate performed better than the N-terminal modified analog. While several parameters of the in vitro experiments were optimized, the overall cell uptake in vitro of radioactivity was still low (<2% of applied radioactivity). Conclusions: A standardized in vitro protocol for evaluating F3 peptide conjugates on cancer cells was established, revealing that the C-terminus is the preferred site for modification. Because the cellular uptake of the radiotracer was shown to likely not be sufficient for radiotracer development, further studies on the optimization of the F3 peptide conjugates, including structural modifications, are required.