It has previously been shown that cholinergic blockade attenuates conditioning-related neuronal responses in human auditory cortex. The present study was conducted to investigate the effect of cholinergic enhancement on such experience-dependent cortical responses. The cholinesterase inhibitor physostigmine, or a placebo control, were continuously infused into healthy young volunteers, during differential aversive conditioning whilst brain activity was measured using event-related functional magnetic resonance imaging (fMRI). Volunteers were presented with two tones, one of which (CS+) was conditioned by pairing with an electrical shock whereas the other was always presented without the shock (CS-). Conditioning-related activations, expressed as an enhanced blood oxygenation level dependent (BOLD) response to the salient CS+, were evident in left auditory cortex under placebo but not under physostigmine. This absence of conditioning-related activations under physostigmine was due to enhanced responses to the CS- under physostigmine as compared to placebo. We suggest that an overactive cholinergic system leads to increased processing of behaviourally irrelevant stimuli and thus attenuates differential conditioning-related cortical activations.