Immuno-PET imaging of the tumor antigen HER2/neu allows for the noninvasive detection and monitoring of oncogene expression; such detection and monitoring are of prognostic value in patients with breast cancer. Compared with the full-size antibody trastuzumab, smaller protein tracers with more rapid blood clearance permit higher imaging contrast at earlier time points. Antigen-binding fragments (Fabs) of antibodies with moderately prolonged circulation achieved through the genetic fusion with a long, conformationally disordered chain of the natural amino acids Pro, Ala, and Ser (PASylation)—a biologic alternative to chemical conjugation with polyethylene glycol, PEG—offer a promising tracer format with improved pharmacokinetics for in vivo imaging. Recently, the transition metal radionuclide ⁸⁹Zr has attracted increasing interest for immuno-PET studies, complementing the conventional halogen radionuclide ¹²⁴I. Methods: To allow direct comparison of these 2 radioactive labels for the same protein tracer, the recombinant αHER2 Fab fused with 200 Pro, Ala, and Ser (PAS₂₀₀) residues was either conjugated with ¹²⁴I via an iodination reagent or coupled with deferoxamine (Df) and complexed with ⁸⁹Zr. After confirmation of the stability of both radioconjugates and quality control in vitro, immuno-PET and biodistribution studies were performed with CD1-Foxn1^nu mice bearing HER2-positive human tumor xenografts. Results: ⁸⁹Zr⋅Df-Fab-PAS₂₀₀ and ¹²⁴I-Fab-PAS₂₀₀ showed specific tumor uptake of 11 and 2.3 percentage injected dose per gram 24 h after injection, respectively; both led to high tumor-to-blood (3.6 and 4.4, respectively) and tumor-to-muscle (20 and 43, respectively) ratios. With regard to off-target accumulation, overt ¹²⁴I activity was seen in the thyroid, as expected, whereas high kidney uptake was evident for ⁸⁹Zr; the latter was probably due to glomerular filtration and reabsorption of the protein tracer in proximal tubular cells. Conclusion: Both ⁸⁹Zr- and ¹²⁴I-labeled versions of αHER2 Fab-PAS₂₀₀ allowed PET tumor imaging with high contrast. With its residualizing radiometal, the tracer ⁸⁹Zr⋅Df-Fab-PAS₂₀₀ showed better in vivo stability and higher tumor uptake.