Morphine infusions into the medial septum produce memory deficits which can be attenuated by concurrent intraseptal injections of glucose. The mnemonic deficits following intraseptal morphine injections may be due, in part, to opioid inhibition of cholinergic neurons projecting to the hippocampus, with glucose reducing the effect. The present experiment determined whether glucose injections into the medial septum attenuate the effects of intraseptal morphine injections on hippocampal acetylcholine release and on memory. Samples of extracellular acetylcholine levels were assessed at 12 min intervals using in vitro microdialysis with high-performance liquid chromatography with electrochemical detection. Intraseptal morphine injections (4.0 nmol) reduced acetylcholine output starting at 12 min and lasting up to 72 min post-injection. Glucose (18.3 nmol) injected concomitantly with morphine reversed the drug infusions in the septum 20 min prior to spontaneous alternation testing. Intraseptal morphine infusions reduced alternation scores; this behavioral effect was reversed by concurrent glucose infusions. The effect of drugs infused into the septal area on spontaneous alternation performance and acetylcholine output were positively correlated. These findings suggest that memory deficits induced by intraseptal morphine injections may result, at least partially, from a decrease in the activity of cholinergic neurons and that this effect is reversed by glucose.