In this overview current insights in the regulation of presynaptic transmitter release, mainly acquired in studies using isolated CNS nerve terminals are highlighted. The following aspects are described. (i) The usefulness of pinched-off nerve terminals, so-called synaptosomes, for biochemical and ultrastructural studies of presynaptic stimulus-secretion coupling. (ii) The regulation of neurotransmitter release by multiple Ca2+ channels, with special emphasis on the specificity of different classes of these channels with respect to the release of distinct types of neurotransmitters, that are often co-localized, such as amino acids and neuropeptides. (iii) Possible molecular mechanisms involved in targeting synaptic vesicle (SV) traffic toward the active zone. (iv) The role of presynaptic receptors in regulating transmitter release, with special emphasis on different glutamate subtype receptors. Isolated nerve terminals are of great value as model system in order to obtain a better understanding of the regulation of the release of distinct classes of neurotransmitters in tiny CNS nerve endings.