Over the last few years several studies have dealt with the importance of the surface charge of nanoparticles in prolonging their blood circulation and minimizing their interaction with plasma proteins. These investigations claimed that zwitterionic nanoparticles exhibited a minimal macrophage response and long blood circulation times compared to nanoparticles with other surface charges. These differences in their in vivo behavior are mainly attributed to the interaction of nanoparticles with plasma proteins. Interestingly, most of these studies considered the total surface charge, instead of the outermost layer of the nanomaterial, as being mainly responsible for these undesirable interactions. However, the first contact with plasma proteins is most likely due to the outermost layer on the nanomaterials. Therefore, here we report a detailed study on the effect of the outermost surface charge of magnetic nanoparticles with regard to biodistribution, pharmacokinetics and bioavailability. Magnetic nanoparticles, coated with PEG chains functionalized with neutral, positive or zwitterionic groups, were intravenously injected into mice, followed in vivo by MRI and then quantified by ICP-MS in blood and the main organs. We found that neutral nanoparticles exhibited long blood circulation times, very good stealth properties and the highest bioavailability, whereas zwitterionic nanoparticles were readily recognized by the mononuclear phagocyte system and avidly taken up by the liver. Also, zwitterionic nanoparticles showed high non-specific cell internalization, whereas neutral nanoparticles showed the lowest cellular uptake, indicating that they require active transport to cross the plasma membrane, which is the desirable situation for therapeutic vehicles with low side effects. Thus, neutral nanoparticles exhibit very favorable characteristics for in vivo applications, whereas zwitterionic nanoparticles show important limitations.