This is a factorial (2 x 2 x 2) spatial memory and cholinergic parameters study in which the factors are chronic ethanol, thiamine deficiency and naivety in Morris water maze task. Both learning and retention of the spatial version of the water maze were assessed. To assess retrograde retention of spatial information, half of the rats were pre-trained on the maze before the treatment manipulations of pyrithiamine (PT)-induced thiamine deficiency and post-tested after treatment (pre-trained group). The other half of the animals was only trained after treatment to assess anterograde amnesia (post-trained group). Thiamine deficiency, associated to chronic ethanol treatment, had a significant deleterious effect on spatial memory performance of post-trained animals. The biochemical data revealed that chronic ethanol treatment reduced acetylcholinesterase (AChE) activity in the hippocampus while leaving the neocortex unchanged, whereas thiamine deficiency reduced both cortical and hippocampal AChE activity. Regarding basal and stimulated cortical acetylcholine (ACh) release, both chronic ethanol and thiamine deficiency treatments had significant main effects. Significant correlations were found between both cortical and hippocampal AChE activity and behaviour parameters for pre-trained but not for post-trained animals. Also for ACh release, the correlation found was significant only for pre-trained animals. These biochemical parameters were decreased by thiamine deficiency and chronic ethanol treatment, both in pre-trained and post-trained animals. But the correlation with the behavioural parameters was observed only for pre-trained animals, that is, those that were retrained and assessed for retrograde retention.