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Flavin mononucleotide (FMN) is a riboflavin derivative that can be exploited to target the riboflavin transporters (RFTs) and the riboflavin carrier protein (RCP) in cells with high metabolic activity. In this study we present the synthesis of different FMN-coated ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) and their efficiency as targeting contrast agents. Since FMN alone cannot stabilize the nanoparticles, we used adenosine phosphates--AMP, ADP and ATP--as spacers to obtain colloidally stable nanoparticles. Nucleotides with di- and triphosphate groups were intended to increase the USPIO charge and thus improve zeta potential and stability. However, all nanoparticles formed negatively charged clusters with similar properties in terms of zeta potential (-28 ± 2 mV), relaxivity (228-259 mM(-1) s(-1) at 3 T) and hydrodynamic radius (53-85 nm). Molecules with a higher number of phosphate groups, such as ADP and ATP, have a higher adsorption affinity towards iron oxide, which, instead of providing more charge, led to partial desorption and replacement of FMN. Hence, we obtained USPIOs carrying different amounts of targeting agent, which significantly influenced the nanoparticles' uptake. The nanoparticles' uptake by different cancer cells and HUVECs was evaluated photometrically and with MR relaxometry, showing that the cellular uptake of the USPIOs increases with the FMN amount on their surface. Thus, for USPIOs targeted with riboflavin derivatives the use of spacers with increasing numbers of phosphate groups does not improve either zeta potential or the particles' stability, but rather detaches the targeting moieties from their surface, leading to lower cellular uptake.