Vitamin A is quite often implicated in supporting acetylcholine synthesis. Choline acetyltransferase, the enzyme promoting acetylcholine synthesis, and the vesicular acetylcholine transporter are modulated by retinoic acid treatment. This paper illustrates the effect of vitamin A deprivation on acetylcholine content in the hippocampus, striatum and prefrontal cortex of rats, brain regions containing retinoid acid receptors. The effect of vitamin A deprivation on working memory was also examined. The results obtained demonstrate a decrease in acetylcholine content following 12 weeks vitamin A deprivation in the hippocampus and striatum, but not in prefrontal cortex. Working memory performance assessed in the same rats was unaffected, suggesting a higher susceptibility of hippocampus and striatum to vitamin A deficiency, in terms of cholinergic transmission.