¹⁸F-Labeled [60]fullerene-based molecular spherical nucleic acids (MSNAs), consisting of a human epidermal growth factorreceptor 2 (HER2) mRNA antisense oligonucleotide sequence with a nativephosphodiester and phosphorothioate backbone, were synthesized, site-specificallylabeled with a positron emitting fluorine-18 and intravenously administratedvia tail vein to HER2 expressing HCC1954 tumor-bearing mice. The biodistributionof the MSNAs was monitored in vivo by positron emissiontomography/computed tomography (PET/CT) imaging. MSNA with a nativephosphodiester backbone (MSNA-PO) was prone to rapid nuclease-mediateddegradation, whereas the corresponding phosphorothioate analogue (MSNA-PS)with improved enzymatic stability showed an interesting biodistributionprofile in vivo. One hour after the injection, majorityof the radioactivity was observed in spleen and liver but also inblood with an average tumor-to-muscle ratio of 2. The prolonged radioactivityin blood circulation may open possibilities to the targeted delivery of the MSNAs.