Hippocampal extracellular acetylcholine (ACh) and choline levels were evaluated using in vivo microdialysis in male Fischer 344 rats before, during, and following an 80-min exposure to two different stress conditions. Measurements were taken in rats restrained and immersed in a water bath containing either 37°C (normothermic-restraint) or 20°C (cold-restraint) water. Results were compared to normothermic-freely-moving rats. Cold- restrained rats displayed decreased ACh levels during cold exposure relative to both normothermic-restrained and normothermic-freely-moving rats. By the end of the cold exposure period and following removal from cold, ACh levels had returned to near-baseline values. Normothermic-restrained rats had levels similar to those of normothermic-freely-moving rats, except for a marked increase in ACh following removal from restraint. Cold-restrained rats displayed a gradual elevation in choline levels during cold stress, followed by a gradual decline after stress termination, whereas both normothermic- restrained and normothermic-freely-moving rats displayed gradual decreases during the microdialysis session. These findings demonstrate that central cholinergic neurotransmission can be altered by the application of, and removal from, acute stressors. In addition, the results suggest a possible relationship between the magnitudes of both the stressor and its cholinergic consequences.