We have recently reported that administration of thin graphene oxide (GO) sheets in the systemic circulation of rodents leads to rapid urinary excretion for the majority of injected dose and accumulation by the reticuloendothelial system organs for the remaining dose. In this study, graphene oxide was functionalized with a chelating moiety (DOTA, (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)) and labeled with [64Cu] for positron emission computed tomography (PET/CT) imaging. The thin functionalized graphene oxide material (f-GO-thin) consisted of a few layers (∼5 nm) in thickness. Aging of the f-GO-thin material led to re-stacking of the flakes that resulted in materials of increased thickness (f-GO-thick) without altering their lateral dimension. These two types of f-GOs were comparatively studied pharmacologically to reveal the previously unexplored in vivo role of graphene oxide sheet thickness. Our results showed that a significantly larger fraction of the thicker GO sheets (47.5% of injected dose) remained within the body of living animals 24 h after intravenous administration, residing mainly in the spleen and liver. The thinner GO sheets were predominantly (76.9% of injected dose) excreted through the glomerular filter into the urine. This pilot study provides an initial correlation between graphene-based material structure and pharmacological profile that is imperative towards understanding of how 2D structures behave in vivo to give information on potential biomedical applications.