Numerous studies have implicated the cholinergic basal forebrain (cBF) in the modulation of auditory cortical responses. This study aimed to accurately define the sources of cBF input to primary auditory cortex (AI) and to assess the efficacy of a cholinergic immunotoxin in cat. Three anaesthetized cats received multiple injections of horseradish-peroxidase conjugated wheatgerm-agglutin into physiologically identified AI. Following one to two days survival, tetramethylbenzidine histochemistry revealed the greatest number of retrogradely labeled cells in ipsilateral putamen, globus pallidus and internal capsule, and smaller numbers in more medial nuclei of the basal forebrain (BF). Concurrent choline acetyltransferase immunohistochemistry showed that almost 80% of the retrogradely labeled cells in BF were cholinergic, with the vast majority of these cells arising from the more lateral BF nuclei identified above. In the second part of the study, unilateral intraparenchymal injections of the cholinergic immunotoxin ME20.4-SAP were made into the putamen/globus pallidus nuclei of six cats. Immuno- and histochemistry revealed a massive reduction in the number of cholinergic cells in and around the targeted area, and a corresponding reduction in the density of cholinergic fibers in auditory cortex. These results are discussed in terms of their implications for investigations of the role of the cBF in cortical plasticity.