Tea tree oil (TTO) is a complex mixture of terpene hydrocarbons. Intensive topical use of TTO in different cosmetics and investigations into its potential as an antimicrobial or anti-inflammatory agent has accentuated the need for studies on the toxicity of TTO. We have applied an experimental in vitro model using static diffusion cells with human skin to study penetration characteristics of terpinen-4-ol and the way TTO affects the barrier integrity of the skin and the percutaneous penetration of two chemicals covering a range of solubilities from 0.03 g/l (methiocarb) to 3.0 g/l (benzoic acid). Through GC-MS analysis we identified the major constituents of TTO. In our experimental set-up with full-thickness skin, only the least lipophilic ingredients of TTO penetrated the skin. Barrier integrity was evaluated through measurement of percutaneous penetration of tritiated water. Data indicate that 1% TTO does not affect barrier conditions. The Kp value for tritiated water was increased significantly at 5% TTO, which demonstrate that the barrier integrity is affected at this relatively low concentration of TTO. The barrier integrity is, however, not seriously damaged, but our data indicate an initiated and concentration-dependent effect on the barrier integrity. TTO changed the penetration characteristics for benzoic acid as well as for methiocarb. The general effect was that TTO reduced the maximal flux. For methiocarb, the lag-time was also prolonged by increasing the TTO concentration in the donor phase to 5%. Thus, TTO reduced the overall amount of benzoic acid as well as methiocarb entering the receptor chamber.