Molecular imaging of tumor antigens using immunospecific magnetic resonance (MR) contrast agents is a rapidly evolving field, which can potentially aid in early disease detection, monitoring of treatment efficacy, and drug development. In this study, we designed, synthetized, and tested in vitro two novel monocrystalline iron oxide nanoparticles (MION) conjugated to antibodies against the her2/neu tyrosine kinase receptor and the 9.2.27 proteoglycane sulfate. MION was synthetized by coprecipitation of iron II and iron III salts in 12-kD dextran solution; antibody coupling was performed by reductive amination. The relaxivity of the conjugates was 24.1-29.1 mM Ϫ1 s Ϫ1 , with 1.8 to 2.1 antibody molecules per nanoparticle. A panel of cultured melanoma and mammary cell lines was used for testing. The cells were incubated with the particles at 16 -32 g Fe/ml in culture medium for 3 h at 37°C, and investigated with immune fluorescence, transmission electron microscopy (TEM), MRI of cell suspensions in gelatine, and spectrophotometric iron determination. All receptor-positive cell lines, but not the controls, showed receptor-specific immune fluorescence, and strong changes in T 2 signal intensity at 1.5 T. The changes in 1/T 2 were between 1.5 and 4.6 s Ϫ1 and correlated with the amount of cell-bound iron (R ϭ 0.92). The relaxivity of cell-bound MION increased to 55.9 Ϯ 10.4 mM Ϫ1 s Ϫ1 . TEM showed anti-9.2.27 conjugates binding to the plasma membrane, while the anti-her2/neu conjugates underwent receptor-mediated endocytosis. In conclusion, we obtained receptor-specific T 2 MR contrast with novel covalently bound, multivalent MION conjugates with anti-9.2.27 and anti-her2/neu to image tumor surface antigens. This concept can potentially be expanded to a large number of targets and to in vivo applications.