The use of nanoparticles as formulation components of topical drug delivery systems for the skin has been widely investigated in the literature. Because of the conflicting conclusions resulting from these studies concerning the ultimate disposition of the nanoparticles employed, the research presented in this paper has been designed to evaluate objectively the fate of such structures when administered to mammalian skin. Confocal microscopy images of skin exposed to nanoparticles have therefore been assessed by quantitative statistical analysis. Sebum on the skin surface was naturally fluorescent and clearly defined the outermost part of the cutaneous barrier. Fluorescent polystyrene nanoparticles applied in aqueous suspension could infiltrate only the stratum disjunctum, i.e., skin layers in the final stages of desquamation. This minimal uptake was independent of contact time (up to 16 h) and of nanoparticle size tested (20–200 nm). When skin barrier function was modestly compromised, the nanoparticles remained incapable of penetration beyond the most superficial layers, corresponding to a depth of 2–3 μm, of the stratum corneum (the outermost, 15–20 μm skin layer). Overall, these results demonstrate objectively and semi-quantitatively that nanoparticles contacting intact, and even partially damaged, skin cannot penetrate beyond the superficial layers of the barrier, and are highly unlikely, therefore, to reach the viable cells of the epidermis or beyond. It follows that nanoparticulate-based, topical delivery systems may prove useful as skin surface reservoirs from which controlled drug release over time may be achieved.