The conventional whole-cell recording of the patch-clamp technique has a great advantage over other electrical recording methods in that it could perfectly control the extra- and intracellular concentrations of ions (such as Na+, K+, Ca2+, and Cl-). However, this advantage is a two-edged sword: it disrupts the physiological ionic environment inside cells. Thus, various Cl(-)-mediated responses in neurons, such as those elicited by inhibitory amino acids, gamma-amino butyric acid (GABA) and glycine (Gly), have been recorded only under an artificial condition. Subsequently, measurement of the normal concentration of intracellular Cl- ([Cl-]i) has been much hampered. [Cl-]i is suggested to play a role in membrane excitability (Deisz and Lux: J Physiol 326: 123-138, 1982) and intracellular signal transduction (Higashijima, Ferguson, and Sternweis: J Biol Chem 262: 3597-3602, 1987), yet the detailed analysis of these fundamental roles has been left undone due to technical difficulties. We have recently developed a "gramicidin perforated patch recording", a novel method of patch-clamp technique that enables analysis of membrane currents with intact [Cl-]i. We here report that by applying this method to acutely dissociated CNS neurons of the rat, we could analyze the Gly-induced membrane currents in the normal ionic environment. And using the reversal potentials, we could deduce the actual [Cl-]i of central neurons.