Summary1. A simple in vitro system was developed to study the effect of y-hydroxybutyrate on nerve cell depolarization-induced release of labelled dopamine, noradrenaline and 5-hydroxytryptamine from brain slices. 2. The release of 3H-dopamine formed in rat striatal slices incubated with 3H-tyrosine was followed either by transferring the slices through successive media or by using a superfusion system. A one to three minute exposure to K+ (53 mM) caused up to a thirty fold increase in the release of newly synthesized 3H-dopamine. This K+-induced release was antagonized when yhydroxybutyrate (1 x 1O-3M) was present in the medium. 3. Potassium (53 mM) increased (eighteen to thirty fold) the release of 3H-dopamine from striatal slices initially loaded by preincubation with 3H-dopamine. However, the K+-induced release of this pool of dopamine was not antagonized by y-hydroxybutyrate. 4. Potassium (53 mM) also increased the release from striatal slices of 3H-5-hydroxytryptamine newly synthesized from 3H-tryptophan. This K+-induced release of 5-hydroxytryptamine was also not inhibited by y-hydroxybutyrate. 5. The release of newly synthesized 3H-noradrenaline from hypothalamic slices was also increased by K+. This K+-induced release, however, unlike that of 5-hydroxytryptamine, was antagonized when y-hydroxybutyrate was present in the superfusion medium. 6. Removal of Ca++ had no effect on K+-induced release of 'H-dopamine when followed by transferring the slices through successive media. This K+-induced release was abolished, however, when Mg++ (12 mM) was present in the medium. 7. The removal of Ca++ from the superfusion medium abolished almost completely the K+-induced release from striatal slices of either newly synthesized 3H-dopamine or preloaded 3H-dopamine. This is presumably due to a more effective washout of tissue Ca++ by the superfusion technique. 8. The ability of y-hydroxybutyrate to antagonize the K+-induced release of monoamines from brain slices does not appear to be unique to the release of newly synthesized dopamine from the striatum.