Two major systems of cholinergic projection neurons are found within the centrum of the mammalian brain: the basal nuclear complex, projecting predominantly to the cerebral cortex, amygdala, and hippocampus, and the pontomesencephalotegmental network, innervating primarily the thalamus. Neurons comprising the latter network also project to the basal forebrain, but the functional properties of that fiber connection, if any, are unknown. In an attempt to address this issue, the extracellular concentration of acetylcholine was measured in the basal nuclear complex of freely moving rats, both singularly and in combination with lesions and pharmacologic manipulations. Acetylcholine release monitored in the presence of physostigmine sulfate in the basal forebrain was (a) calcium-dependent, (b) increased by systemic scopolamine injection, the rise persisting in the presence of quisqualate lesions of the basal nuclear complex, (c) blocked by tetrodotoxin, and (d) abolished by ablation of cell bodies in the pontomesencephalic tegmentum, which also produced a decrease of choline acetyltransferase activity in the nucleus basalis/substantia innominata region, but not by quisqualate lesions of the basal forebrain. It is concluded from these data that the calcium-dependent release of acetylcholine in the basal nuclear complex (a) is largely axonal in nature, (b) derives substantially from axons of the cholinergic pontomesencephalic tegmentum, and (c) appears to be controlled by presynaptic muscarinic receptors on axon terminals of the latter system. The pontomesencephalotegmental cholinergic complex might thus influence cortical acetylcholine release, in part at least, by means of serial-order cholinergic-cholinergic interactions in the basal nuclear complex.