Although several dextran-coated iron oxide preparations are in preclinical and clinical use, little is known about the mechanism of uptake into cells. As these particles have been shown to accumulate in macrophages and tumor cells, we performed cellular uptake and inhibition studies with a prototypical monocrystalline iron oxide nanoparticle (MION). MION particles were labeled with fluorescein isothiocyanate or radioiodinated and purified by gel permeation chromatography. Two preparations of MION particles were used in cell experiments: nontreated MION and plasma-opsonized MION purified by gradient density purification. As determined by immunoblotting, opsonization resulted in C3, vitronectin, and fibronectin association with MION. Incubation of cells with fluorescent MION showed active uptake of particles in macrophages both before and after opsonization. In C6 tumor cells, however, intracellular MION was only detectable in dividing cells. Quantitatively, 125I-labeled MION was internalized into cells with uptake values ranging from 17 ng (in 9L gliosarcoma) to 970 ng iron per million cells for peritoneal macrophages. Opsonization increased MION uptake into macrophages sixfold, whereas it increased the uptake in C6 tumor cells only twofold. Results from uptake inhibition assay suggest that cellular uptake of nonopsonized (dextran-coated) MION particles is mediated by fluid-phase endocytosis, whereas receptor-mediated endocytosis is presumably responsible for the uptake of opsonized (protein-coated) particles.