Unilateral stereotaxic injection of 3.5 nmol of kainic acid into the ventral globus pallidus of rats reduced biochemical cholinergic neuronal markers by 45-50% and virtually eliminated histochemical staining for acetylcholinesterase in neocortex ipsilateral to the lesion. At the lesion site, the large, multipolar neurons that stain densely for acetylcholinesterase were absent when compared with the uninjected side. Kainate was as effective as electrocoagulation for reducing cholinergic markers although it did not affect aminergic projections ascending through the lesioned area. The conclusion that the cholinergic projection originated in neuronal perikarya at the lesion site was supported by the failure of kainate or electrolytic lesions in contiguous regions to produce similar effects. These studies provide strong evidence for a cholinergic projection to neocortex from neurons in the forebrain in the nucleus basalis.The mammalian cerebral cortex contains the neurotransmitter acetylcholine (AcCho) and moderately high levels of activity of its biosynthetic enzyme choline acetyltransferase (acetylCoA:choline O-acetyltransferase; EC 2.3.1.6) and its catabolic enzyme acetylcholinesterase (AcCho-esterase; acetylcholine acetylhydrolase; EC 3.1.1.7) (1, 2). AcCho and drugs that alter cholinergic neurotransmission influence cortical electrical activity, and under physiologic conditions AcCho is released from the surface of the neocortex (3). Neocortical neurons, especially in the deeper layers, are strongly excited by the iontophoretic application of AcCho (4); and muscarinic cholinergic receptors have been characterized in neocortex by neurophysiologic and neurochemical methods (5-7). Ligand binding studies with a-bungarotoxin have also demonstrated the presence of nicotinic receptors in cortex (8, 9). These findings suggest that the neocortex receives a substantial cholinergic innervation.Previous studies on the possible source of cortical cholinergic innervation have, however, been inconclusive. Undercutting of the cerebral cortex to lesion cortical afferents results in a marked reduction in the activity of choline acetyltransferase, suggesting a subcortical source of cholinergic innervation (10); however, interpretation of the effects of this local lesion is confounded by the possible direct or retrograde damage to cortical intrinsic cholinergic neurons. Shute and Lewis, in a series of elegant experiments, used histochemical techniques to stain for AcCho-esterase activity in neuronal fibers and concluded that the neocortical cholinergic innervation arose from perikarya situated in the brain stem and diencephalon (11); however, the recent demonstration that many noncholinergic neurons are also cholinesterase-positive has undermined the specificity of their technique (12-14). In contrast, McGeer et al. (15), utilizing antiserum against choline acetyltransferase to stain for neurons containing this enzyme, which presumably are cholinergic, reported that choline acetyltransferase was confined to cortical intrins...