A technique has been developed to measure 3H-GABA not only in tissues but also in medium of slices of the rat substantia nigra (SN) incubated for 15 min with 3H-glutamine. The quantity of 3H-GABA in tissues was about 30 to 35 times that released in the medium. Nevertheless, the amount of the 3H-transmitter spontaneously released was about 10 to 15 times the blank value. GAD activity in the SN was decreased by 40 and 80% respectively ten days after the kainic acid lesion of the ipsilateral striatum or hemitransection. These effects were associated with parallel reductions in the amounts of 3H-GABA accumulated in tissues and released in medium. The spontaneous release of newly synthesized 3H-GABA was increased in absence of calcium and reduced with an excess of calcium (10 −2M). Tetrodotoxin (10 −5, 5.10 −6M) reduced by 40% the spontaneous release of 3H-GABA. These various effects were not associated with significant change in the total accumulation of 3H-GABA in tissues + medium. Finally depolarization of the slices with potassium (30 mM) increased the release of 3H-GABA (300%). This effect was abolished in absence of calcium and was not associated with a significant change in the amount of 3H-GABA accumulated in tissues.