The effects of contextual fear conditioning on the release of acetylcholine (ACh) in the hippocampus of freely moving rats was assessed using microdialysis. Measures were carried out during both acquisition and retention testing (re-exposure to the conditioning chamber) and compared between animals that either received foot-shocks as unconditioned stimulus (conditioned group) or no foot-shocks (control group) during acquisition. Results showed that during acquisition, hippocampal ACh extracellular level was increased with respect to baseline but that this increase was of similar magnitude in both groups. By contrast, re-exposure to the conditioning chamber the day after (retention testing) produced a significantly greater increase in ACh extracellular level in the conditioned (that, otherwise, displayed conditioned freezing behavior to contextual cues), than in the control group (which displayed virtually no freezing). This enhanced hippocampal ACh release seems to result from the greater hippocampal processing of contextual stimuli in conditioned animals with respect to controls.