The extent and duration of cholinergic hypofunction induced by long-term ethanol consumption was investigated in the rat. Ethanol (20% v/v) was administered to male adult Wistar rats as a sole source of fluid for three or six months. Control rats received tap water. The body weight, food and fluid intake in ethanol-treated rats were lower than in control rats throughout the treatment. After three months of ethanol consumption, and one week withdrawal, acetylcholine release in freely moving rats, investigated by microdialysis technique coupled to high-performance liquid chromatography quantification, was significantly decreased by 57 and 32% in the hippocampus and cortex, respectively, while choline acetyltransferase activity was significantly decreased (-30%) only in the hippocampus. A complete recovery of choline acetyltransferase activity and acetylcholine release was found after four ethanol-free weeks. Conversely, after four weeks of withdrawal following six months of ethanol treatment, the recovery in acetylcholine release was not accompanied by that in choline acetyltransferase activity, which remained significantly lower than in control rats in both cortex and hippocampus. The ability of rats to negotiate active and passive avoidance conditioned response tasks, tested after four ethanol-free weeks, was strongly impaired in both three- and six-month ethanol-treated rats. In conclusion, our experiments demonstrate that the development of a long-lasting cholinergic hypofunction requires at least six months of ethanol administration. The hypofunction affects choline acetyltransferase activity and acetylcholine release differently, and undergoes a remarkable recovery.