Because of the relative scarcity of fresh human oral mucosa specimens for permeability studies, we investigated the use of human vaginal mucosa as a model of the former. In a previous study we demonstrated the comparable diffusion rate of water across human vaginal and buccal mucosa and proposed the use of the former as a suitable model of the latter for in vitro drug permeability studies. To further evaluate the human vaginal/buccal mucosa model, we decided to compare these two tissues with respect to their permeability to 17β-estradiol. Clinically healthy human vaginal and buccal mucosa specimens were obtained during vaginal hysterectomies and different oral surgical procedures. The permeability of each tissue specimen to 17β-estradiol was determined through the use of a continuous flow-through diffusion system. Specimens were examined histologically before and after experiments. Mean flux values for 17β-estradiol across human buccal mucosa tended to be slightly higher than those observed for vaginal tissue, but no statistically significant differences could be demonstrated. The results from this study further support our hypothesis that human vaginal mucosa may be a suitable model of human buccal mucosa for in vitro drug permeability studies.