Phenserine (PS) was designed as a selective acetylcholinesterase (AChE) inhibitor, with a tartrate form (PST) for oral administration in mild to moderate Alzheimer's disease (AD). Recent phase 3 trials of PST in Europe indicate that any clinically relevant activity of PST may be limited by its duration of action. Like many oral drugs, bioavailability and plasma concentrations of PST are regulated by hepatic and gastrointestinal first-pass effects. To minimize the kinetic limitations of first-pass metabolism, transdermal formulations of PS and PST (ointment/patch) were developed and characterized in vitro and in vivo. Initial in vitro kinetic characterization of PS or PST formulations used a diffusion cell chamber and skin samples isolated from hairless mice. Liquid paraffin and fatty alcohol/propylene glycol (FAPG) were found to be suitable vehicles for ointment formulation. Addition of a penetration enhancer, 1-[2-(decylthio)ethyl]-azacyclopentane-2-one (HPE-101), improved stratum corneum permeability. Application of the optimal formulation of PS/HPE-101/FAPG to the shaved back of rats resulted in significantly lowered plasma and brain AChE activities and improved cognitive performance in animals with scopolamine-induced cognitive impairment. These results suggest that the transdermal application of AChE inhibitors may represent an effective therapeutic strategy for AD. Particular benefits over oral therapies might include avoiding first-pass metabolic effects and improved dosing compliance.