The present study was designed to evaluate the release of acetylcholine in the prefrontal cortex (PFC) induced by handling stress during aging and also to investigate whether this response changed as a result of the animals living in an enriched environment. Male Wistar rats of 3 months of age were housed in control and enriched conditions during the entire period of their adult life and experiments were performed at 6, 15 and 24 months of age. Spontaneous motor activity was first monitored in an open field arena. Then, rats were stereotaxically implanted with guide cannula to perform microdialysis experiments in the PFC and to evaluate the effects of stress on extracellular concentrations of acetylcholine. Handling stress increased the extracellular concentrations of acetylcholine in the PFC of control and enriched rats. These increases were not modified by aging in control rats. However, environmental enrichment (EE) reduced the effects of stress on acetylcholine concentrations in all groups of age. Spontaneous motor activity in the open field was reduced by aging. EE also decreased motor activity in all groups of age. These results suggest that EE reduces the reactivity to stress of the cholinergic system in the prefrontal cortex during aging.