Breast cancer (BC) consists of multiple subtypes defined by various molecular characteristics, for instance, estrogen receptor (ER) expression. Methods for visualizing BC include mammography, MR imaging, ultrasound, and nuclear medicine-based methods such as (99m)Tc-sestamibi and (18)F-FDG PET, unfortunately all lacking specificity. Peptide receptor scintigraphy and peptide receptor radionuclide therapy are successfully applied for imaging and therapy of somatostatin receptor-expressing neuroendocrine tumors using somatostatin receptor radioligands. On the basis of a similar rationale, radioligands targeting the gastrin-releasing peptide receptor (GRP-R) might offer a specific method for imaging and therapy of BC. The aim of this study was to explore the application of GRP-R radioligands for imaging and therapy of BC by introducing valid preclinical in vitro and in vivo models. Methods: GRP-R expression of 50 clinical BC specimens and the correlation with ER expression was studied by in vitro autoradiography with the GRP-R agonist (111)In-AMBA. GRP-R expression was also analyzed in 9 BC cell lines applying (111)In-AMBA internalization assays and quantitative reverse transcriptase polymerase chain reaction. In vitro cytotoxicity of (177)Lu-AMBA was determined on the GRP-R-expressing BC cell line T47D. SPECT/CT imaging and biodistribution were studied in mice with subcutaneous and orthotopic ER-positive T47D and MCF7 xenografts after injection of the GRP-R antagonist (111)In-JMV4168. Results: Most of the human BC specimens (96%) and BC cell lines (6/9) were found to express GRP-R. GRP-R tumor expression was positively (P = 0.026, χ(2)(4) = 12,911) correlated with ER expression in the human BC specimens. Treatment of T47D cells with 10(-7) M/50 MBq of (177)Lu-AMBA resulted in 80% reduction of cells in vitro. Furthermore, subcutaneous and orthotopic tumors from both BC cell lines were successfully visualized in vivo by SPECT/CT using (111)In-JMV4168; T47D tumors exhibited a higher uptake than MCF7 xenografts. Conclusion: Targeting GRP-R-expressing BC tumors using GRP-R radioligands is promising for nuclear imaging and therapy, especially in ER-positive BC patients.