Cortical acetylcholine (ACh) release has been linked to various cognitive functions, including perceptual learning. We have previously shown that cortical cholinergic innervation is necessary for accurate sound localization in ferrets, as well as for their ability to adapt with training to altered spatial cues. To explore whether these behavioral deficits are associated with changes in the response properties of cortical neurons, we recorded neural activity in the primary auditory cortex (A1) of anesthetized animals in which cholinergic inputs had been reduced by making bilateral injections of the immunotoxin ME20.4-SAP in the nucleus basalis (NB). The pattern of spontaneous activity of A1 units recorded in the ferrets with cholinergic lesions (NB ACh–) was similar to that in controls, although the proportion of burst-type units was significantly lower. Depletion of ACh also resulted in more synchronous activity in A1. No changes in threshold, frequency tuning or in the distribution of characteristic frequencies were found in these animals. When tested with normal acoustic inputs, the spatial sensitivity of A1 neurons in the NB ACh– ferrets and the distribution of their preferred interaural level differences also closely resembled that found in control animals, indicating that these properties had not been altered by sound localization training with one ear occluded. Simulating the animals’ previous experience with a virtual earplug in one ear reduced the contralateral preference of A1 units in both groups, but caused azimuth sensitivity to change in slightly different ways, which may reflect the modest adaptation observed in the NB ACh– group. These results show that while ACh is required for behavioral adaptation to altered spatial cues, it is not required for maintenance of the spectral and spatial response properties of A1 neurons.