The effect of 3,3',5-triiodo-L-thyronine (T3) on working memory in ethylcholine aziridinium ion (AF64A)-treated mice was studied in a delayed non-matching to sample task using a T-maze. After behavioural testing was completed, mice were killed by microwave irradiation and regional brain levels of acetylcholine, aspartate, glutamate, glutamine, glycine, taurine, and gamma-aminobutyric acid (GABA) were measured by high-performance liquid chromatography with electrochemical detection. Treatment with AF64A (7 nmol, i.c.v.) produced a deficit in working memory performance in the non-matching to sample task at 30 s delay, and decreased acetylcholine, glutamate, and GABA levels in the hippocampus, but not in the septum and cerebral cortex. Administration of T3 (0.3 mg/kg, p.o., once daily for 6 days) to AF64A-treated animals improved the deficit in working memory performance and reversed the decrease in acetylcholine, glutamate, and GABA levels in the hippocampus. These results indicate that the deficit in performance induced by AF64A can be improved by T3 administration.