The cholinergic system is known to show deterioration during aging and Alzheimer's disease (AD). In response, a therapeutic approach to AD has been to attempt to compensate for the decrease in central cholinergic function by potentiating the activity of the remaining intact cholinergic cells with cholinesterase (ChE) inhibitors. In this study treatment with the long-lasting ChE inhibitor metrifonate facilitated acquisition and retention of eyeblink conditioning in aging rabbits. Metrifonate treatment resulted in steady-state, dose-dependent acetylcholinesterase (AChE) inhibition in red blood cells. Maximal behavioral efficacy was achieved with AChE inhibition of approximately 40%, with no further improvements resulting from increased levels of inhibition. Metrifonate was behaviorally effective in the absence of the severe side effects that can plague ChE inhibitors, supporting metrifonate as a possible treatment for the cognitive deficits resulting from normal aging and AD.