The permeation enhancing property of 5% oleic acid in ethanol on beta-estradiol was investigated in vitro and in vivo using symmetrical and asymmetrical side-by-side diffusion cells and the human skin sandwich flap, respectively. beta-Estradiol permeability in vitro and in vivo was similar in 75% ethanol (ETOH). Oleic acid (5%) did not alter beta-estradiol permeability in vivo but increased permeability six-fold in vitro in symmetrical diffusion cells. beta-Estradiol permeability in oleic acid was not different from that in ETOH, however, using asymmetrical diffusion cells. Stratum corneum-to-vehicle partition coefficients of beta-estradiol in the vehicles were similar, yet fourfold more steroid was detected in skin biopsies from the in vitro symmetrical diffusion cells. Thus, oleic acid increased beta-estradiol permeability in vitro only when skin was equilibrated with fatty acid. Attention to in vitro diffusion cell design and its relevance in vivo is critical to defining the mechanisms of enhanced solute permeation.