Background As a tomographic imaging technology, magnetic particle imaging (MPI) allows high spatial resolution and sensitivity, and the possibility to create real-time images by determining the spatial distribution of magnetic particles. To ensure a prospective biosafe application of UL-D (University of Luebeck-Dextran coated superparamagnetic nanoparticles), we evaluated the biocompatibility of superparamagnetic iron oxide nanoparticles (SPIONs), their impact on biological properties, and their cellular uptake using head and neck squamous cancer cells (HNSCCs). Methods SPIONs that met specific MPI requirements were synthesized as tracers. Labeling and uptake efficiency were analyzed by hematoxylin and eosin staining and magnetic particle spectrometry. Flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays, and real-time cell analyzer assays were used to investigate apoptosis, proliferation, and the cytokine response of SPION-labeled cells. The production of reactive oxygen species (ROS) was determined using a fluorescent dye. Experimental results were compared to the contrast agent Resovist ® , a standard agent used in MPI. Results UL-D nanoparticles and Resovist particles were taken up in vitro by HNSCCs via unspecific phagocytosis followed by cytosolic accumulation. To evaluate toxicity, flow cytometry analysis was performed; results showed that dose- and time-dependent administration of Resovist induced apoptosis whereas cell viability of UL-D-labeled cells was not altered. We observed decreased cell proliferation in response to increased SPION concentrations. An intracellular production of ROS could not be detected, suggesting that the particles did not cause oxidative stress. Tumor necrosis factor alpha (TNF-α) and interleukins IL-6, IL-8, and IL-1β were measured to distinguish inflammatory responses. Only the primary tumor cell line labeled with >0.5 mM Resovist showed a significant increase in IL-1β secretion. Conclusion Our data suggest that UL-D SPIONs are a promising tracer material for use in innovative tumor cell analysis in MPI.