The classically conditioned rabbit nictitating membrane reflex (NMR) is modulated by the septohippocampal cholinergic system. Disruption of this system retards NMR acquisition. Aluminium (Al) is a neurotoxin that interferes with hippocampal acetylcholine (ACh) synthesis and release. Using microdialysis, this study tested the hypothesis that NMR acquisition in the rabbit is associated with hippocampal ACh release. This was conducted by measuring ACh release in control and A1-intoxicated rabbits during NMR training. NMR training consisted of four sessions of 100 conditioning trials/session in a delay paradigm. The percentage of conditioned responses (CRs) increased with each conditioning session for both groups, although percent CRs was significantly greater in the control group. Acetylcholine release in the ventral hippocampus increased significantly over baseline in the control group during the second and third conditioning sessions. In the Al-intoxicated group, ACh release did not increase significantly during any conditioning session. A separate group of rabbits was pseudoconditioned, receiving the same conditioning stimuli, although explicitly unpaired. This group did not acquire the CR. Acetylcholine release did not significantly increase during any conditioning session, suggesting that the increase in ACh release observed in the control group was not merely a product of conditioning stimuli presentation. The lack of increased ACh release in the Al-intoxicated rabbits was associated with a CR acquisition deficit. The results of this study are consistent with a role of hippocampal cholinergic function in NMR acquisition in the rabbit.