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Because brain membranes contain large amounts of docosahexaenoic acid (DHA, 22:6n-3), and as (n-3) PUFA dietary deficiency can lead to impaired attention, learning, and memory performance in rodents, we have examined the influence of an (n-3) PUFA-deprived diet on the central cholinergic neurotransmission system. We have focused on several cholinergic neurochemical parameters in the frontal cortex and hippocampus of rats fed an (n-3) PUFA-deficient diet, compared with rats fed a control diet. The (n-3) PUFA deficiency resulted in changes in the membrane phospholipid compositions of both brain regions, with a dramatic loss (62–77%) of DHA. However, the cholinergic pathway was only modified in the hippocampus and not in the frontal cortex. The basal acetylcholine (ACh) release in the hippocampus of deficient rats was significantly (72%) higher than in controls, whereas the KCl-induced release was lower (34%). The (n-3) PUFA deprivation also caused a 10% reduction in muscarinic receptor binding. In contrast, acetylcholinesterase activity and the vesicular ACh transporter in both brain regions were unchanged.Thus, we evidenced that an (n-3) PUFA-deficient diet can affect cholinergic neurotransmission, probably via changes in the phospholipid PUFA composition.