Metrifonate is currently under development as a putative cholinergic Alzheimer therapeutic, because it is a prodrug of the long-acting organophosphate cholinesterase (ChE) inhibitor dichlorvos. The aim of this study was to examine whether the transformation of metrifonate to dichlorvos and the resulting indirect inhibition of ChE are required for its previously documented cognition-enhancing properties in a standard Morris water escape task with intact rats. This was done by investigating whether the cognition-enhancing effects of metrifonate could be mimicked by dichlorvos, by the organophosphorus compounds diisopropylfluorophosphate and paraoxon or by structurally unrelated reference ChE inhibitors, such as tetrahydroaminoacridine, E2020, and physostigmine. Metrifonate, and to a lesser degree dichlorvos, and diisopropylfluorophosphate improved the acquisition of the water escape task, whereas paraoxon did not. The dose-response curves of the organophosphorus compounds were bell-shaped with apparent optimal doses of 10 to 30 mg/kg for metrifonate and 0.03 mg/kg for both dichlorvos and diisopropylfluorophosphate. The reference compounds E2020, physostigmine and tetrahydroaminoacridine did not affect learning and memory in the young-adult rat at doses that had previously been reported to mediate cognitive enhancement in deficiency models. Our results question whether the effect of metrifonate is mediated by inhibition of ChE alone and suggest the involvement of an additional, as yet unknown, mechanism of action.