Recent immunoelectron microscopic studies have revealed a low frequency of synaptic membrane differentiations on ACh (ChAT-immunostained) axon terminals (boutons or varicosities) in adult rat cerebral cortex, hippocampus and neostriatum, suggesting that, besides synaptic transmission, diffuse transmission by ACh prevails in many regions of the CNS. Cytological analysis of the immediate micro-environment of these ACh terminals, as well as currently available immunocytochemical data on the cellular and subcellular distribution of ACh receptors, is congruent with this view. At least in brain regions densely innervated by ACh neurons, a further aspect of the diffuse transmission paradigm is envisaged: the existence of an ambient level of ACh in the extracellular space, to which all tissue elements would be permanently exposed. Recent experimental data on the various molecular forms of AChE and their presumptive role at the neuromuscular junction support this hypothesis. As in the peripheral nervous system, degradation of ACh by the prevalent G4 form of AChE in the CNS would primarily serve to keep the extrasynaptic, ambient level of ACh within physiological limits, rather than totally eliminate ACh from synaptic clefts. Long-lasting and widespread electrophysiological effects imputable to ACh in the CNS might be explained in this manner. The notions of diffuse transmission and of an ambient level of ACh in the CNS could also be of clinical relevance, in accounting for the production and nature of certain cholinergic deficits and the efficacy of substitution therapies.