BACKGROUND: Iron oxide nanoparticles have been approved by food and drug administration for clinical application as magnetic resonance imaging (MRI) and are considered to be a biocompatible material. Large iron oxide nanoparticles are usually used as transversal (T) contrast agents to exhibit dark contrast in MRI. In contrast, ultrasmall iron oxide nanoparticles (USPIONs) (several nanometers) showed remarkable advantage in longitudinal (T)-weighted MRI due to the brighten effect. The study of the toxicity mainly focuses on particles with size of tens to hundreds of nanometers, while little is known about the toxicity of USPIONs. RESULTS: We fabricated FeO nanoparticles with diameters of 2.3, 4.2, and 9.3 nm and evaluated their toxicity in mice by intravenous injection. The results indicate that ultrasmall iron oxide nanoparticles with small size (2.3 and 4.2 nm) were highly toxic and were lethal at a dosage of 100 mg/kg. In contrast, no obvious toxicity was observed for iron oxide nanoparticles with size of 9.3 nm. The toxicity of small nanoparticles (2.3 and 4.2 nm) could be reduced when the total dose was split into 4 doses with each interval for 5 min. To study the toxicology, we synthesized different-sized SiO and gold nanoparticles. No significant toxicity was observed for ultrasmall SiO and gold nanoparticles in the mice. Hence, the toxicity of the ultrasmall FeO nanoparticles should be attributed to both the iron element and size. In the in vitro experiments, all the ultrasmall nanoparticles (< 5 nm) of FeO, SiO, and gold induced the generation of the reactive oxygen species (ROS) efficiently, while no obvious ROS was observed in larger nanoparticles groups. However, the ·OH was only detected in FeO group instead of SiO and gold groups. After intravenous injection, significantly elevated ·OH level was observed in heart, serum, and multiple organs. Among these organs, heart showed highest ·OH level due to the high distribution of ultrasmall FeO nanoparticles, leading to the acute cardiac failure and death. CONCLUSION: Ultrasmall FeO nanoparticles (2.3 and 4.2 nm) showed high toxicity in vivo due to the distinctive capability in inducing the generation of ·OH in multiple organs, especially in heart. The toxicity was related to both the iron element and size. These findings provide novel insight into the toxicology of ultrasmall FeO nanoparticles, and also highlight the need of comprehensive evaluation for their clinic application.