Macromolecules such as proteins are attracting increasing interest for molecular imaging. We previously proposed a novel strategy for preparing macromolecules labeled with a PET radionuclide, C-11, using a cell-free translation system with C-11-methionine. However, macromolecules tend to exhibit slower kinetics, thus requiring a longer scanning time. Here, we expand our strategy using F-18, which has a longer half-life, with the cell-free translation system with 4-F-18-fluoro-L-proline (F-18-FPro). We evaluated F-18-interleukin-8 (F-18-IL-8) produced by this method in vitro and in vivo to provide a proof of concept of our strategy. Methods: We tested some fluorinated amino acids to be incorporated into a protein. Trans-F-18-FPro was radiolabeled from the corresponding precursor. F-18-IL-8 was produced using the cell-free translation system with trans-F-18-FPro instead of natural L-proline with incubation at 37 degrees C for 120 min. An in vitro binding assay of F-18-IL-8 was performed using IL-8 receptor-expressing cells. After intravenous administration of F-18-IL-8, in vivo PET imaging of IL-8 receptor-expressing xenograft-bearing mice was performed using a small-animal PET system. Results: FPro was identified as an amino acid incorporated into the protein. F-18-IL-8 was successfully prepared using the cell-free translation system and trans-F-18-FPro with the radiochemical yield of 1.5% (decay-corrected) based on trans-F-18-FPro. In vitro binding assays of F-18-IL-8 demonstrated its binding to IL-8 receptor. In vivo PET imaging demonstrated that F-18-IL-8 clearly accumulated in IL-8 receptor-expressing xenografts in mice, unlike trans-F-18-FPro. Conclusion: F-18-IL-8 produced by this method binds to IL-8 receptors in vitro, and F-18-IL-8 PET clearly visualizes its target receptor-expressing xenograft in vivo. Therefore, this technique might be useful for labeling macromolecules and performing preclinical evaluations of proteins of interest in vitro and in vivo.