Humectants belong to a group of hydrophilic compounds frequently used in skin care products with the aim to diminish the clinical symptom of skin dryness. The biochemical and biophysical mechanisms by which humectants interact with the skin barrier are far from fully understood. Increased understanding of such mechanisms can enhance the possibilities to tailor skin care products for various skin abnormalities.The work presented in this thesis centres on one high (hyaluronan) and two low (urea and glycerol) molecular weight humectants and their interactions with water, as well as their effect on the barrier properties of the outermost layer of the skin, i.e. the stratum corneum (SC). We explore the effect of hydration on thermodynamic properties of humectants, in particular hyaluronan, by using isothermal sorption calorimetry, differential scanning calorimetry and small- and wide-angle X-ray scattering. By combining data from several methods, a binary phase diagram of the hyaluronan - water system was constructed.We also investigate the effect of hydration and presence of humectants on the SC permeability in vitro by using an experimental set-up that allows for control of the boundary conditions in terms of water activity. In contrast to low molecular weight humectants, like urea and glycerol, it was concluded that hyaluronan (17 kDa) does not penetrate the skin barrier due to size exclusion. Addition of urea, glycerol or hyaluronan to aqueous formulations inevitably lowers the water activity of the formulation, which in tum affects the SC permeability when being applied. Moreover, it was shown that skin permeability of a model drug metronidazole decreases upon addition of hyaluronan to the formulation, while high skin permeability was maintained with addition of urea or glycerol. In addition, skin membrane electrical resistance, which normally increases at dehydrating skin conditions, remained low in presence of urea and glycerol.Excised skin hydrated at different hydration levels were examined with confocal Raman microspectroscopy. Large water inclusions were observed in fully hydrated SC after 24h exposure to a buffer solution. Addition of urea was shown to promote the formation of these inclusions. Urea and glycerol were also shown to improve the hydration capacity of isolated comeocytes.Similar approach as used in vitro was employed in vivo to explore the effect of hydration and humectants on skin permeability. It was shown that the water activity of the applied formulations have a marked effect on the barrier properties and urea and glycerol was shown to improve skin hydration even at reduced water activity of the applied formulation.