Affibody molecules are small (7 kDa), non-immunoglobulin scaffold proteins with a potential as targeting agents for radionuclide imaging of cancer. However, high renal re-absorption of Affibody molecules prevents their use for radionuclide therapy with residualizing radiometals. We hypothesized that the use of Affibody-based peptide nucleic acid (PNA)-mediated pretargeting would enable higher accumulation of radiometals in tumors than in kidneys. To test this hypothesis, we designed an Affibody-PNA chimera Z_HER2:342-SR-HP1 containing a 15-mer HP1 PNA recognition tag and a complementary HP2 hybridization probe permitting labeling with both ¹²⁵I and ¹¹¹In. ¹¹¹In-Z_HER2:342-SR-HP1 bound specifically to HER2-expressing BT474 and SKOV-3 cancer cells in vitro, with a K_D of 6±2 pM for binding to SKOV-3 cells. Specific high affinity binding of the radiolabeled complementary PNA probe ¹¹¹In-/¹²⁵I-HP2 to Z_HER2:342-SR-HP1 pre-treated cells was demonstrated. ¹¹¹In-Z_HER2:342-SR-HP1 demonstrated specific accumulation in SKOV-3 xenografts in BALB/C nu/nu mice and rapid clearance from blood. Pre-saturation of SKOV-3 with non-labeled anti-HER2 Affibody or the use of HER2-negative Ramos xenografts resulted in significantly lower tumor uptake of ¹¹¹In-Z_HER2:342-SR-HP1. The complementary PNA probe ¹¹¹In/¹²⁵I-HP2 accumulated in SKOV-3 xenografts when Z_HER2:342-SR-HP1 was injected 4 h earlier. The tumor accumulation of ¹¹¹In/¹²⁵I-HP2 was negligible without Z_HER2:342-SR-HP1 pre-injection. The uptake of ¹¹¹In-HP2 in SKOV-3 xenografts was 19±2 %ID/g at 1 h after injection. The uptake in blood and kidneys was approximately 50- and 2-fold lower, respectively. In conclusion, we have shown that the use of Affibody-based PNA-mediated pretargeting enables specific delivery of radiometals to tumors and provides higher radiometal concentration in tumors than in kidneys.