Designed ankyrin repeat proteins (DARPins) are genetically engineered proteins that exhibit high specificity and affinity toward specific targets. Here, the G3-DARPin, which binds the HER2/neu receptor, was site-specifically modified with enzymatic methods and 89Zr-radiolabeled for applications in positron emission tomography (PET). Sortase A transpeptidation was used to install a desferrioxamine B (DFO) chelate bearing a reactive triglycine group to the C-terminal sortase tag of the G3-DARPin, and 89Zr-radiolabeling produced a novel 89ZrDFO-G3-DARPin radiotracer that can detect HER2/neu-positive tumors. The triglycine probe, DFO-Gly3 (1), was synthesized in 29% overall yield. After sortase A transpeptidation and purification from the nonfunctionalized protein component, the DFO-G3-DARPin product was radiolabeled to give 89ZrDFO-G3-DARPin. Binding specificity was assessed in HER2/neu-expressing BT-474 and SK-OV-3 cellular assays. The pharmacokinetics, tumor uptake, and specificity of 89ZrDFO-G3-DARPin were measured in vivo by PET imaging and confirmed by final time point (24 h) biodistribution experiments in female athymic nude mice bearing BT-474 xenografts. Sortase A transpeptidation afforded the site-specific and stoichiometrically precise functionalization of DFO-G3-DARPin with one chelate per protein. The modified DFO-G3-DARPin was purified from the nonfunctionalized DARPin by using Ni-NTA affinity chromatography. 89ZrDFO-G3-DARPin was obtained with a radiochemical purity of >95% measured by radio-size-exclusion chromatography. BT-474 tumor uptake at 24 h postadministration reached 4.41 ± 0.67 %ID/g (n = 3) with an approximate ∼70% reduction in tumor-associated activity in the blocking group (1.26 ± 0.29 %ID/g; 24 h postadministration, n = 5, P-value of <0.001). Overall, the site-specific, enzyme-mediated functionalization and characterization of 89ZrDFO-G3-DARPin in HER2/neu positive BT-474 xenografts demonstrate that DARPins are an attractive platform for generating a new class of protein-based radiotracers for PET. The specific uptake and retention of 89ZrDFO-G3-DARPin in tumors and clearance from most background tissues produced PET images with high tumor-to-background contrast.