1. By the development of a new perforated patch method using gramicidin, the effects of GABA on neurones dissociated from the rat substantia nigra pars reticulata (SNR) were examined without disturbing the intracellular chloride concentration. 2. Using the patch pipette solution containing gramicidin (100 micrograms ml-1), the access resistance dropped to less than 20 M omega within 40 min after making the gigaohm seal. 3. Under current-clamp conditions, GABA caused a hyperpolarization accompanied by a blockade of spontaneous firing. Under voltage clamp at a holding potential (Vh) of -50 mV, GABA evoked an outward current by way of bicuculline- and picrotoxin-sensitive GABAA receptors. 4. A 10-fold change of extracellular chloride concentration resulted in a 58 mV shift of the reversal potential of GABA-induced outward current (EGABA), indicating that the membrane behaves like a chloride electrode in the presence of GABA. 5. The intracellular chloride activities (aCli), calculated with the Nernst equation using both extracellular chloride activity and EGABA values, ranged from 2.8 to 19.7 mM with a mean value of 9.5 mM. The aCli was not affected either by different pipette solutions or by different holding potentials more hyperpolarized than -40 mV. 6. In the recording from SNR neurones in brain slice using the gramicidin-perforated patch-clamp technique, the inhibitory and excitatory postsynaptic currents were recorded in different current directions and the former was blocked by bicuculline. 7. In conclusion, the gramicidin-perforated patch method will disclose previously unknown aspects of biological responses involving Cl-.