Acetylcholine (ACh) was collected from the alvear surface of the dorsal hippocampus and cerebral cortex in chloralose-urethane anaesthetized or unanaesthetized rabbits. With anaesthesia, the resting release of ACh from the hippocampus was greater than that from the cortex. Wthout anaesthesia, the resting release from both areas was much higher and very similar. The addition of atropine sulphate (1 microgram/ml) to the collecting fluid or the administration of Artane (2 mg/kg i.v.) increased resting ACh release from both the hippocampus and cortex to similar output levels. Atropine also increased ACh release due to stimulation of the medial septum (MS) or mesencephalic reticular formation (MRF). Removal of the septum abolished the effect of atropine on resting ACh release and on release evoked by MRF stimulation from both the hippocampus and cortex. The data indicate that the septum is an essential pathway for cholinergic fibres ascending to the cerebral cortex and hippocampus. They also demonstrate that the septal cholinergic fibres must be intact and active for atropine to increase ACh release from their terminals.