Twelve sesquiterpene compounds, derived from natural volatile oils, were investigated as putative skin penetration enhancers for human skin. Pretreatment of epidermal membranes with sesquiterpene oils, or solid sesquiterpenes saturated in dimethyl isosorbide, increased the rate of absorption of the model hydrophilic permeant, 5-fluorouracil (5-FU). Enhancers with polar functional groups were generally more potent than pure hydrocarbons. Furthermore, enhancers with the least bunched structures were the most active. The largest effect was observed following pretreatment with nerolidol, which increased pseudo-steady-state 5-FU flux over 20-fold. Molecular modelling suggested that terpenes with structures suitable for alignment within lipid lamellae were the most potent enhancers. Sesquiterpene enhancers had long durations of action implying that they did not wash out of the skin easily. This study attempted to improve enhancer clearance by replacing the aqueous donor and receptor phases by ethanol:water (1:1) solutions. Ethanol increased the permeability coefficient for 5-FU 13-fold, demonstrating that, in aqueous solution, it is a moderately potent penetration enhancer. Sesquiterpene and ethanol enhancement effects were approximately additive. Sesquiterpene effects were almost fully maintained for at least 4.5 days following pretreatment, illustrating poor reversibility. Stratum corneum/water drug partitioning studies suggested that an important mechanism of action of the enhancers was to increase the apparent drug diffusivity in the stratum corneum. Increases in drug partitioning into the entire stratum corneum following enhancer pretreatment were relatively small. Diffusivity increases were directly related to overall rises in permeability. This study has shown that sesquiterpene compounds, which are of low toxicity and cutaneous irritancy, can promote 5-FU absorption across human skin. Sesquiterpene compounds, therefore, show promise as clinically-acceptable skin penetration enhancers.