BACKGROUND: The aim of this study was to produce ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (NPs) conjugated to the FROP-1 peptide for targeted magnetic resonance imaging (MRI) of breast cancer cell lines and to evaluate its application as a specific and targeted T1-weighted MR imaging contrast agent in vitro. Sodium citrate-stabilized FeO NPs were conjugated with the FROP-1 peptide by 1-ethyl-3-(3-dimethylaminopropyl) carbide diamide hydrochloride (EDC) to form a novel FeO@FROP-1 specific target contrast agent. The specificity and targeting of FeO@FROP-1 to bind FROP-1 receptors were investigated in vitro by cellular uptake and cellular MR imaging. RESULTS: In this study, the synthesis of water-soluble ultrasmall FeO NPs was performed by the co-precipitation method. XRD, TEM, and VSM analyses showed the formation of the FeO NPs with an average size of about 3.78 ± 0.2 nm. FT-IR spectroscopy approved the conjugation of the FROP-1 peptide with the FeO NPs. The synthesized FeO@FROP-1 NPs showed good biocompatibility, and the high r1 relaxivity and r2/r1, respectively, were 2.608 mMS and 1.18. The biocompatibility of the FeO and FeO@FROP-1 NPs on the MCF-7, SKBR-3, MDA-MB-231, and MCF-10 cell lines was determined using cytotoxicity analysis. The specific targeting effect on the cells was verified by in vitro cellular uptake and cell MR imaging. CONCLUSION: It was found that the contrast intensity of the FeO@FROP-1 nanoprobe increases as Fe concentration increases. Cellular uptake of the FeO and FeO@FROP-1 NPs was quantified using ICP-MS. The synthesized NPs had better imaging performance than Dotarem (gadoterate meglumine). The findings showed that FeO@FROP-1 NPs have potential utility as a specific and targeted T1-weighted contrast agent in breast cancer MR imaging.