1. Measurements of brain acetylcholine (ACh) synthesis from precursor [14C]-pyruvate, pantothenic acid (PA) concentration in the brain, and blood ethanol (EtOH) concentration were made in rats treated with either ethanol (5-6 g kg-1 body wt daily) alone or ethanol with PA supplementation (100-200 mg kg-1 body wt daily). EtOH with or without PA was administered orally by either Lieber-Decarli liquid diet for 4 weeks and 4 months or by oral intubation for 1 and 4 days. Matched controls were given either ethanol-free liquid diet or saline. 2. ACh synthesis in the brain of rats treated with ethanol alone for 4 months was significantly (P less than 0.01) inhibited. PA concentration of the brain was diminished to 7.0% of the control value. 3. PA concentration in the brain of rats treated with ethanol plus PA for 4 months was three times that of rats treated with ethanol alone. ACh synthesis in rats with ethanol and PA supplementation was also significantly (P less than 0.01) higher. 4. There was no difference in blood EtOH concentration between rats treated with ethanol with or without PA supplement. 5. The EtOH effect on ACh synthesis and PA concentration in the brain was observed in the chronic treatments but not in the acute treatments. 6. Data suggest that chronic ethanol exposure may decrease ACh synthesis by depleting PA, a precursor for the synthesis of acetyl CoA. Acetyl CoA is an essential substrate for ACh synthesis.