<b><i>Background and Aim:</i></b> Investigations on chemical enhancement of skin penetration of gold nanoparticles are considered crucial to have a deeper insight into the main barrier of particle penetration. <b><i>Methods:</i></b> In this study, penetration of gold nanoparticles in the presence of several chemical enhancers – urea, sodium lauryl sulphate, polysorbate 80 and dimethyl sulfoxide (DMSO) – through human skin was studied. <b><i>Results:</i></b> Among the tested chemical enhancers, DMSO could induce the penetration of hydrophilic (citrate-stabilized) gold colloid of no intrinsic penetration ability in a concentration-dependent manner. Pretreatment of the skin with DMSO however reduced the penetration of hydrophobic (cetrimide-coated) gold nanoparticles as a result of aggregation in the top layers of the stratum corneum limiting penetration into the deeper skin layers. In addition, nanoparticles-vehicle interaction and the stability of the nanoparticles in the applied vehicle were found important determinants of skin penetration. <b><i>Conclusion:</i></b> Our results demonstrate that the already established approaches for chemical permeation enhancement of drug molecules and their postulated mechanisms could be used as preliminary guidelines for enhancing the penetration of nanoparticles. At this size range of 15 nm, intercellular lipids provide the main barrier to particle penetration through the stratum corneum.