The cholinergic system has been implicated in sensory cortical plasticity, learning and memory. This experiment determined the relationship between the acquisition of a Pavlovian conditioned approach response (CR) to an auditory conditioned stimulus (CS) and the release of acetylcholine (ACh) in the primary auditory cortex in rats. Samples of ACh were collected via microdialysis during behavioral training in either an auditory classical conditioning task or in a non-associative control task. The conditioning group received daily pairings of a white noise CS with a sucrose pellet unconditioned stimulus (US), while the control group received an equal number of CS and US presentations, but with these stimuli being presented randomly. Training was conducted on three consecutive days, with microdialysis samples being collected on Days 1 and 3 in separate sub-groups. The level of ACh released in the auditory cortex during conditioning trials increased from the first to the third day of training in the conditioning group as rats acquired the CR, but did not change in the control group, which did not acquire a CR. These data provide direct evidence for the hypothesis that ACh release increases in the primary auditory cortex during natural memory formation, where cholinergic activation is known to contribute to the formation of specific associative representational plasticity in conjunction with specific memory formation.