Cell labeling with various types of nanomaterial, such as FDA-approved iron oxide nanoparticles (IONPs) has become common practice in biomedical research. The low uptake of IONPs stimulates the use of transfection agents (TA), but the effect on stability of the IONPs and their cellular interactions has received minimal attention. In the present study, we evaluated the use of Lipofectamine as a commonly used TA and tested different ratios of TA and IONPs. While the TA–IONP complexes are stable in saline, at a high ratio of TA over IONP, substantial aggregation occurred in serum-containing media. Even for the highest ratio, TA was unable to completely cover the IONPs, resulting in a net negative charge of all complexes. At high TA–IONP ratios, more complexes remained surface-associated without internalization, resulting in cell death, while at lower TA–IONP ratios, complexes were more avidly taken up through fluid-phase pinocytosis and clathrin-mediated endocytosis. At later time points, the endocytosed complexes accumulated within the lysosomes and affected the appearance of lysosomal structures. The data indicate that TAs should be used with care as, depending on the ratio of TA and IONP, the complexes may aggregate, inducing cell death and preventing internalization. Copyright © 2012 John Wiley & Sons, Ltd.