The in vivo effects of fatty acids on skin barrier function were assessed by measuring: (i) transepidermal water loss (TEWL), (ii) diffusion lag times for hexyl nicotinate (HN), and (iii) irritant skin response using laser Doppler velocimetry (LDV) in combination with visual scoring. Two classes of fatty acids have been investigated: straight-chain saturated fatty acids (SFA), having 6–12 carbon atoms, and unsaturated fatty acids (UFA): oleic, linoleic, α-linolenic and arachidonic acids. It has been reported that these acids can enhance the permeation of various compounds across the skin. After topical and occlusive application as a solution in propylene glycol (PG) for 3 h on the volar arm of human subjects, SFA only caused a slight irritation and increase in TEWL. The diffusion lag times of HN were reduced by the application SFA to the same extent as and not more than by the application of the pure solvent PG. In contrast, the application of UFA caused a significant increase in TEWL and LDV (irritation) responses. The TEWL values after oleic acid application were higher than those observed for the other three acids, while the irritation potential of arachidonic acid was the highest among UFA. As with SFA, sites treated with UFA did not show significantly different lag times of HN diffusion from PC-treated sites. The data suggest that the degree of irritation and the degree of barrier modulation for fatty acids are not necessarily correlated.