Effects of acute and chronic phenobarbital treatment on GABA, glutamate, aspartate, glycine and taurine contents in seven regions of the rat brain have been investigated. Rats treated chronically with phenobarbital maintained the hepatic changes known to follow short term treatment and high phenobarbital levels in the liver, brain and serum. After withdrawal, spontaneous convulsions were first observed when phenobarbital level in the serum fell to about 15 micrograms/ml. Chronic phenobarbital treatment resulted in the decrease of GABA content in the cerebral cortex, midbrain, hypothalamus, striatum and hippocampus. At 2 d after withdrawal, glutamate and aspartate contents were decreased and taurine content was increased with recovery of GABA content. Acute phenobarbital treatment resulted in the decrease of GABA and glutamate contents and the increase of taurine content. The changes of amino acid contents were different among seven regions of the rat brain, but phenobarbital distributed uniformly after acute and chronic treatment. These results indicate that GABA participates, at least in part, in phenobarbital action and abstinence syndrome may arise from a deficiency of GABA.