The safety of topical application of Australian tea tree Oil (TTO) is confounded by a lack of transdermal penetration data, which adequately informs opinions and recommendations. In this study we applied TTO in its pure form and as a 20% solution in ethanol in vitro to human epidermal membranes from three different donors, mounted in horizontal Franz-type diffusion cells, using normal 'in use' dosing conditions (10 mg/cm2). In addition, we examined the effect of partially occluding the application site on the penetration of TTO components. Our data showed that only a small quantity of TTO components, 1.1-1.9% and 2-4% of the applied amount following application of a 20% TTO solution and pure TTO, respectively, penetrated into or through human epidermis. The largest TTO component penetrating the skin was terpinen-4-ol. Following partial occlusion of the application site, the penetration of terpinen-4-ol increased to approximately 7% of the applied TTO. Measurement of the rate of evaporation of tea tree oil from filter paper (7.4 mg/cm2) showed that 98% of the oil evaporated in 4 hours. Overall, it is apparent that the penetration of TTO components through human skin is limited.