Rats were trained to press a lever under a 'Multiple FI-60s and Extinction' schedule with food reinforcements. After learning the task, an in vivo microdialysis probe was inserted into the dentate gyrus and CA3 regions of the hippocampus, and the sequential changes in the dialysate acetylcholine (ACh) concentration were analyzed. In the session, two fixed-interval of reinforcement (FI) components (for 20 min) and two extinction (EXT) components (for 30 min) were alternated to examine the correlation between behavioral and neurochemical outcomes. The dialysate ACh concentration increased during the FI component and returned to the baseline during the EXT component of the schedule. Next, in order to dissociate the effect of discrimination from the effect of rewarding on the neurochemical changes in the hippocampus, we used a final TEST period (for 20 min) during which the actual schedule was extinction but the discriminative stimulus was on, i.e. the manifest condition of the test period was reinforcement. In the TEST period, the animals pressed the lever with almost the same frequency as during the FI component; however, the dialysate ACh concentration did not increase above the baseline concentration. These results suggest that ACh release in the rat hippocampus is associated with reinforcement but not with discrimination in operant conditioning.