In this study, the overflow of acetylcholine (ACh) in the septo-hippocampal system was studied using intracerebral microdialysis in freely moving rats. Dialysis probes were implanted in the ventral hippocampus and in the medial septal area (MS), including a part of the ventral limb of the diagonal band of Broca (VDB). Dialysis samples were analysed 'on-line' using HPLC with post column enzymatic conversion and electrochemical detection. Local perfusion of 1 mumol/l of the sodium-channel blocker tetrodotoxin (TTX) through the probe resulted in 94% and 92% decrease in extracellular levels of ACh in the hippocampus and the septal area, respectively. The effects of septal manipulation on the efflux of ACh in the hippocampus were studied by electrical stimulation of the septal area and by administering drugs via the septal probe. Electrical stimulation of the MS/VDB caused a 336% increase in the output of ACh in the hippocampus. Perfusion of 3 mumol/l TTX through the septal probe caused a maximal decrease of 56% in the output of ACh in the ventral hippocampus. When perfused in the MS/VDB, the excitatory amino-acid agonists N-methyl-D-aspartate (NMDA) (100 mumol/l) and kainic acid (10 mumol/l) caused an increase in the extracellular level of ACh in the hippocampus by 83% and 161%, respectively. Thus, the overflow of ACh in the hippocampus and the septal area both depend on neuronal impulse flow. The extracellular level of ACh in the hippocampus is at least partially dependent on impulse flow in septo-hippocampal fibres. Moreover, the output of ACh in the hippocampus can be manipulated by electrical and pharmacological stimulation of the MS/VDB.