Rats received a solution of sodium barbital as their only drinking fluid for 25 and 30 weeks. Four groups were studied: (1) control; (2) barbital until sacrifice; (3) barbital withheld (abstinent) for 3 days; (4) abstinent for 12 days. Abstinence convulsions in groups 3 and 4 were recorded with jiggle cages. The rats were killed by decapitation and the concentration of acetylcholine (ACh) was measured in 3 parts of the brain: striatum, hippocampus + cerebral cortex, cerebellum + medulla oblongata + midbrain. In animals receiving barbital until sacrifice, no significant change in ACh content was found in any of the brain regions compared with controls. In animals abstinent for 3 days and with a maximal frequency of spontaneous convulsions a decreased content of ACh (--35%) was found in the striatum. On the 12th day of abstinence, when the convulsive activity clearly had decreased, the ACh content was still decreased (--30%) in the striatum and a significant decrease compared with controls was also found in the cerebellum + medulla oblongata + midbrain.