Acetylcholine (ACh) is one of the neurotransmitters in the CNS and associated with various types of physiological phenomena, such as arousal state, learning and memory, Alzheimer disease and epilepsy. At present, ACh concentration in vivo can be monitored using enzymatic electrodes and microdialysis techniques. In the present study, we developed a new ion-selective electrode to detect ACh concentration, and applied it to the brain. The electrode was high sensitivity, because its lower limit of detection was 3 Χ 10^-7 M ACh. The electrode was also selective, because interference by typical inorganic ion was negligible. Next, we attempted to identify cholinergic pathways in the brain, by use of the ACh-selective electrode. It is known that the supramammillary nucleus (SuM) of the hypothalamus regulates neural activity in the hippocampus. We have also reported that the SuM regulates temporal lobe epilepsy in a model of hippocampal sclerosis. However, neurotransmitters in the neural pathway from the SuM to the hippocampus are unknown. We therefore examined changes in the concentration of ACh in the hippocampus in response to stimulation of the SuM, using the ACh electrode. Administration of saline to the SuM as a control did not induce changes in the concentration of ACh in the hippocampus. On the other hand, administration of muscimol, a GABA_A receptor agonist, to the SuM remarkably increased the concentration of ACh in the hippocampus. These results show that there is a cholinergic pathway from the SuM to the hippocampus, and also that the newly developed electrode is capable of applying to the brain.