Male Sprague-Dawley rats were bilaterally adrenalectomized and corticosterone (CORT) was substituted as subcutaneous pellets in two groups of animals: low- (L-CORT: 1 x 25 mg pellet) or high-level of CORT (H-CORT: 4 x 100 mg pellet). Between 14 and 19 days after CORT substitution, ethylcholine aziridinium (AF64A) was intracerebroventricularly (i.c.v.) injected in the CORT long-term exposed rats and the dose- and time-dependent effect of this treatment was measured on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in hippocampus and septum and on serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and noradrenaline (NA) levels in hippocampus. Rats were killed at 2, 4, 7 and 14 days after AF64A treatment. Starting 4 days after the i.c.v. administration of 0.5 or 1.0 nmol of AF64A, an aggravation of the reduction of ChAT activity was measured in the hippocampus of the H-CORT animals compared to the L-CORT ones. In the septum of the H-CORT rats, the activity of ChAT increased within the first week after the infusion of the toxin, while no significant effect was observed in the L-CORT group. As we observed with ChAT, AF64A induced a severe inhibition of AChE activity in the hippocampus of the H-CORT rats compared to the L-CORT ones. In the septum, an increase of AChE activity was observed in both groups of CORT-exposed animals. In the hippocampus of H-CORT animals, the exacerbation of the inhibition of ChAT and AChE activity was accompanied by a parallel decrease in the content of 5-HT and 5-HIAA starting 4 days after AF64A injections. Finally, NA content in hippocampus was not affected by the toxin in the CORT-substituted animals. These data demonstrate that: (1) long-term exposure to supraphysiological levels of CORT enhances the cholinodisruption induced in hippocampus by AF64A, at doses of 0.5 and 1.0 nmol/side; (2) high circulating plasma CORT concentrations impair hippocampal cholinergic neuronal capacity to recover from damage; and (3) the degree of inhibition of the serotoninergic system is augmented in H-CORT animals, most probably due to an adaptation of the serotoninergic neurons to the larger withdrawal of cholinergic function observed in this group.