Ultrasmall nanoparticles (US-NPs; <20 nm in hydrodynamic size) are now included in a variety of pharmacological and cosmetic products, and new technologies are needed to detect at high sensitivity the passage of small doses of these products across biological barriers such as the skin. In this work, a diffusion cell adapted to positron emission tomography (PET), a highly sensitive imaging technology, was developed to measure the passage of gold NPs (AuNPs) in skin samples in continuous mode. US-AuNPs (3.2 nm diam.; TEM) were functionalized with deferoxamine (DFO) and radiolabeled with 89Zr(IV) (half-life: 3.3 days, matching the timeline of diffusion tests). The physicochemical properties of the functionalized US-AuNPs (US-AuNPs-PEG-DFO) were characterized by FTIR (DFO grafting; hydroxamate peaks: 1629.0 cm–1, 1569.0 cm–1), XPS (presence of the O═C–N C 1s peak of DFO at 287.49 eV), and TGA (organic mass fraction). The passage of US-AuNPs-PEG-DFO-89Zr(IV) in skin samples was measured by PET, and the diffusion parameters were extracted thereby. The signals of radioactive US-AuNPs-PEG-DFO-89Zr(IV) leaving the donor compartment, passing through the skin, and entering the acceptor compartment were detected in continuous at concentrations as low as 2.2 nM of Au. The high-sensitivity acquisitions performed in continuous allowed for the first time to extract the lag time to the start of permeation, the lag time to start of the steady state, the diffusion coefficients, and the influx data for AuNPs permeating into the skin. PET could represent a highly valuable tool for the development of nanoparticle-containing topical formulations of drugs and cosmetics.Read less <