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This study investigated the relationship between spatial working memory and hippocampal long-term potentiation (LTP) using the allocentric place discrimination task (APDT) in rats, in which the selection accuracy is a good index for spatial working memory. Either the selective M1 muscarinic receptor antagonist pirenzepine (50 μg) or the choline uptake inhibitor hemicholinium-3 (5 μg) impaired APDT selection accuracy, but neither affected the induction of LTP in the hippocampal CA1 region in anesthetized rats. In contrast, the selective N-methyl-D-aspartate receptor antagonist D-amino-5-phosphonopentanoate (200 nmol) did not impair APDT selection accuracy but completely blocked hippocampal CA1 LTP. These results suggest that spatial working memory is independent of hippocampal CA1 LTP and that the central cholinergic system is involved in spatial working memory, but not through the modulation of hippocampal CA1 LTP.