This study examined the effects of N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate on basal and electrically-evoked release of acetylcholine (ACh) from the rat hippocampal and striatal slices which were preincubated with [3H]choline. Unexpectedly, the basal and evoked ACh release were not affected at all by the treatment with NMDA (3∼100μM), AMPA (1∼100μM) or kainate (1∼100μM) in hippocampal slices. However, in striatal slices, under the Mg2⁢-free medium, 30μM NMDA increased the basal ACh release with significant decrease of the electrically- evoked releases. The treatment with 1μM MK-801 not only reversed the 30μM NMDA-induced decrease of the evoked ACh release, but also attenuated the facilitatory effect of 30μM NMDA on the basal ACh release. The treatment with either 30μM AMPA or 100μM kainate increased the basal ACh release without any effects on the evoked release. The treatment with 10μM NBQX abolished the AMPA- or kainate-induced increase of the basal ACh release. Interestingly, NBQX significantly attenuated the evoked release when it was treated with AMPA, although it did not affect the evoked release alone without AMPA. These observations demonstrate that in hippocampal slices, ionotropic glutamate receptors do not modulate the ACh release in cholinergic terminals, whereas in striatal slices, activations of ionotropic glutamate receptors increase the basal ACh release though NMDA may decrease the electrically-evoked ACh release.