The ethidium bromide (EB) demyelinating model was associated with interferon beta (IFN-beta) to evaluate acetylcholinesterase (AChE) activity in the striatum (ST), hippocampus (HP), cerebral cortex (CC), cerebellum (CB), hypothalamus (HY), pons (PN) and synaptosomes from the CC. Rats were divided into four groups: I control (saline), II (IFN-beta), III (EB) and IV (EB and IFN-beta). After 7, 15 and 30 days rats (n = 6) were sacrificed, and the brain structures were removed for enzymatic assay. AChE activity was found to vary in all the brain structures in accordance with the day studied (7-15-30 days) (P < 0.05). In the group III, there was an inhibition of the AChE activity in the ST, CB, HY, HP and also in synaptosomes of the CC (P < 0.05). It was observed that IFN-beta per se was capable to significantly inhibit (P < 0.05) AChE activity in the ST, HP, HY and synaptosomes of the CC. Our results suggest that one of the mechanisms of action of IFN-beta is through the inhibition of AChE activity, and EB could be considered an inhibitor of AChE activity by interfering with cholinergic neurotransmission in the different brain regions.