Single-domain antibody fragments (sdAbs) are promising vectors for immuno-PET; however, better methods for labeling sdAbs with F are needed. Herein, we evaluate a site-specific strategy using an F residualizing motif and the anti-epidermal growth factor receptor 2 (HER2) sdAb 5F7 bearing an engineered C-terminal GGC tail (5F7GGC). 5F7GGC was site-specifically attached with a tetrazine-bearing agent via thiol-maleimide reaction. The resultant conjugate was labeled with F by inverse electron demand Diels-Alder cycloaddition with a -cyclooctene attached to 6-F-fluoronicotinoyl moiety via a renal brush border enzyme-cleavable linker and a PEG chain (F-5F7GGC). For comparisons, 5F7 sdAb was labeled using the prototypical residualizing agent, -succinimidyl 3-(guanidinomethyl)-5-I-iodobenzoate (-I-SGMIB). The 2 labeled sdAbs were compared in paired-label studies performed in the HER2-expressing BT474M1 breast carcinoma cell line and athymic mice bearing BT474M1 subcutaneous xenografts. Small-animal PET/CT imaging after administration of F-5F7GGC in the above mouse model was also performed. F-5F7GGC was synthesized in an overall radiochemical yield of 8.9% ± 3.2% with retention of HER2 binding affinity and immunoreactivity. The total cell-associated and intracellular activity for F-5F7GGC was similar to that for coincubated -I-SGMIB-5F7. Likewise, the uptake of F-5F7GGC in BT474M1 xenografts in mice was similar to that for -I-SGMIB-5F7; however, F-5F7GGC exhibited significantly more rapid clearance from the kidney. Small-animal PET/CT imaging confirmed high uptake and retention in the tumor with very little background activity at 3 h except in the bladder. This site-specific and residualizing F-labeling strategy could facilitate clinical translation of 5F7 anti-HER2 sdAb as well as other sdAbs for immuno-PET.