Estradiol transdermal application still represents a relevant topic due to the recent controversies about hormone replacement therapy, its increasing potential in the treatment of skin aging and wound healing, and the new use of its derivatives in the transdermal contraceptive treatment. The aim of our work was to verify if fresh or frozen rabbit ear skin can be a suitable skin model to study in vitro estradiol transdermal absorption. Fresh rabbit ear skin demonstrated a reasonable model of human epidermis in the investigation of estradiol permeation starting from both solutions and commercial patches.