5-HT4 receptor (5-HT4R) activation increases memory and learning. As the loss of cholinergic markers is one of the major neurochemical deficits in Alzheimer's disease (AD), an increase in acetylcholine (ACh) release by 5-HT4R activation may be of therapeutic benefit to alleviate symptoms in AD. The facilitation of ACh release from hippocampal brain slices by 5-HT4R activation has been described by Siniscalchi et al. (1999). We used this method to evaluate the influence of novel selective 5-HT4R agonists on hippocampal ACh release. The hippocampus was dissected from rat brain. Hippocampal slices were cut at 350 mm, loaded with [³H]-choline, and transferred to 200 ml chambers (perfused with carbogen-saturated Krebs' solution containing hemicholinium-3 (2.10–6 M); flow rate of 1.2 ml/min). After 1 hour of perfusion, 3 min samples of the effluent were collected and assayed for tritium content (by scintillation counting) as a measure of [³H]-ACh . Electrical field stimulation (biphasic pulses, 1 ms duration, 50 mA, 2 Hz, for 2 min) was applied at 15 and 45 min after beginning the collection (S1, S2); drugs were added 15 min before S2. In accordance with literature, atropine (10–6 M) increased and the adenosine receptor agonist CADO (10–5 M) decreased the tritium release by 36% and 55%, respectively (p<0.001 vs. control). This corresponds with the blockade and stimulation of presynaptic inhibitory control on ACh release through muscarinic and adenosine receptors respectively. However, neither BIMU-8 (2.10–6 M), the 5-HT4R agonist that was reported by Siniscalchi to increase tritium efflux by 43%, nor the highly selective 5-HT4R agonist prucalopride (10–6 M) increased tritium outflow. In the present study, selective 5-HT4R agonists do not facilitate ACh release from hippocampal brain slices. This might be related to the model system (ex vivo) as the facilitatory effect of 5-HT4R activation on hippocampal ACh release was shown in vivo (Matsumoto, 2001, JPET).