Because brain extracellular acetylcholine (ACh) levels are near detection limits in microdialysis samples, an acetylcholinesterase (AChE) inhibitor such as neostigmine is often added to microdialysis perfusates to increase ACh levels in the dialysate, a practice that raises concerns that the inhibitor might alter the results. Two experiments compared functional differences in ACh release with and without neostigmine. In the first experiment, 30–60% increases in extracellular ACh concentrations in the hippocampus were evident during food-rewarded T-maze training with 20–500 nM neostigmine in the perfusate but no increases were seen without neostigmine. In the second experiment, 78% increases in ACh release in the hippocampus were seen after injections of the GABAA receptor antagonist, bicuculline, into medial septum only if neostigmine (50 nM) was included in the perfusate. These findings suggest that, in the hippocampus, endogenous brain AChEs are very efficient at removing extracellular ACh, obscuring differences in ACh release in these experiments. Therefore, inclusion of AChE inhibitors in the microdialysis perfusate may be necessary under some conditions for observations of functional changes in release of ACh in the hippocampus.