Reliable noninvasive methods are needed to monitor cell engraftment and graft survival after hepatocyte transplantation. Superparamagnetic iron oxide nanoparticles (SPIOs) have been shown to accumulate in various types of cells, and are currently the labeling agent of choice for cellular magnetic resonance imaging (MRI). However, for successful clinical translation to hepatocyte transplantation, it is important that hepatocytes maintain their viability and synthetic function after labeling. In this study, primary human hepatocytes were incubated with increasing concentrations of clinical grade SPIOs for different time intervals. SPIOs uptake was confirmed by light and fluorescence microscopy, and intracellular iron content quantified by a colorimetric ferrozine-based assay. Studies were performed to determine if labeling affected cell viability and function. Intracellular iron concentrations increased in a time- and dose-dependent manner after incubation with SPIOs. Labeling had minimal short-term effects on cell attachment and mitochondrial function. However, exposure of hepatocytes to SPIOs resulted in a dose- and time-dependent reduction in protein synthesis. Cell labeling for 16 h had no significant effect on hepatocyte-specific function, but longer periods of incubation resulted in a dose-dependent decrease in albumin production. Hepatocytes incorporated SPIOs at sufficient levels for in vitro detection on a 7-T MRI imaging system, with a minimum of 2,000 SPIO-labeled cells/μl detected by a decreased T2 relaxivity compared to controls. Intrasplenic transplantation of human hepatocytes labeled with 50 μg Fe/ml of SPIOs was performed in nonobese diabetic/severe combined immune deficiency (NOD-Scid) mice. Recipient livers showed a clear decrease in signal intensity on T2*-weighted MR images when compared to controls, allowing detection of hepatocytes. With further experiments to optimize the conditions for labeling human hepatocytes, it should be possible to apply this technique to track hepatocyte transplantation in patients with liver disease.