Ferumoxtran, an ultrasmall superparamagnetic iron oxide particle, can be located in several tissue compartments in the liver, namely the extracellular space (blood and interstitium), reticuloendothelial cells, and possibly hepatocytes. To better understand the compartmental distribution of ferumoxtran in the liver, we performed a longitudinal study in the rat using microscopy and magnetic resonance imaging. At light microscopy, no substantial cellular uptake of ferumoxtran was observed before one hour after injection. With a dose of 15 micromol Fe/kg, the number of ferumoxtran particles in the reticuloendothelial cells peaked between one and four hours and with a 150 micromol Fe/kg dose, it peaked between eight and 24 hours. Within hepatocytes, only sparse particles were observed with electron microscopy, at a dose of 150 micromol Fe/kg. Imaging performed up until one hour after ferumoxtran injection showed a significant increase in liver signal intensity on T1-weighted images. These results suggest that ferumoxtran mainly acts as an extracellular agent for at least one hour in the rat and that reticuloendothelial accumulation peaks at later time points. Substantial uptake within hepatocytes did not occur.