y-Butyrolactone, a depressant drug of the central nervous system, has been investigated for its ability to alter brain levels of 5-hydroxytryptamine, y-aminobutyric acid and acetylcholine in mice and rats; of these three compounds, only acetylcholine was changed in amount. Levels of acetylcholine in the cerebral cortex were increased by y-butyrolactone with a time-course which closely followed the depressed state of the animal. Indirect evidence has been presented to show that in the mid-brain and brain stem the change in acetylcholine level induced by y-butyrolactone is sharply localized in an area of the mesencephalon that contains the corpora quadrigemina.