We applied the perforated-patch-clamp technique to cultured cortical neurons of the rat to characterize the ionic basis of membrane potential changes and membrane currents induced by gamma-aminobutyric acid (GABA). Gramicidin was used as the membrane-perforating agent, to allow the recording of whole-cell currents without impairing the intracellular Cl- concentration ([Cl-]i). In current-clamp experiments in the presence of 26 mM HCO3- the application of 50 microM GABA evoked changes in the membrane potential of neurons including depolarizations (19%), hyperpolarizations (38%) and biphasic changes in membrane potential (31%), characterized by a transient hyperpolarization followed by a sustained depolarization. Accordingly, GABA (50-200 microM) induced inward, outward or biphasic current responses under voltage-clamp. Inward and biphasic currents as well as depolarizations and biphasic membrane potential responses, respectively, occurred more frequently in the presence of 26 mM HCO3-. The second phase of the biphasic membrane potential or current responses was markedly reduced when the preparation was bathed in a HCO3--free saline, indicating a contribution from HCO3-. The reversal potential of the GABA-induced currents (EGABA) determined with the gramicidin-perforated-patch mode and in the nominal absence of HCO3- was -73 mV, while it was shifted to -59 mV in the presence of HCO3-. Combined patch-clamp and microfluorimetric measurements using the Cl--sensitive dye 6-methoxy-1-(3-sulphonatopropyl)quinolinium (SPQ) showed that GABA evoked an increase of [Cl-]i in 54% (n=13) of the neurons. We conclude that this increase of [Cl-]i in combination with the efflux of HCO3- results in a shift of EGABA above the resting membrane potential that gives rise to GABA-mediated depolarizations.