gamma-Aminobutyric acid (GABA), the most common inhibitory neurotransmitter in the vertebrate brain, acts by increasing the conductance of the neuronal membrane to chloride ions. The addition of GABA to monolayer cultures of chick cerebral neurons produced a 3-fold increase in the uptake of 36Cl-. This stimulation was maximal during the first 20 s after GABA addition but declined rapidly thereafter. The GABA-dependent uptake activity was doubled by increasing the external K+ concentration from 5.5 to 40 mM. The dependence of the 36Cl- entry rate on the external concentrations of GABA (K0.5 = 6 microM; Vmax = 4.4 nmol/mg of cell protein/s) and Cl-(Km = 105 mM; Vmax = 9.9 nmol mg-1 s-1) followed Michaelis-Menten kinetics. The GABA analog, muscimol, produced a similar response (K0.5 = 8 microM; Vmax = 5.2 nmol mg-1 s-1). While 50 microM 3-aminopropane sulfonate also stimulated 36Cl- uptake, 2,4-diaminobutyrate, taurine, and glycine were without effect. Bicuculline (Ki = 3.5 microM) was a noncompetitive inhibitor of GABA-dependent Cl- entry, while the inhibition by picrotoxin (Ki = 1.0 microM) was uncompetitive with GABA. Nearly one-half of the basal activity, observed in the absence of GABA, was blocked by the anion transport inhibitors, furosemide or 4-acetamido-4'-isothiocyano-2,2'-stilbene sulfonate, but these compounds gave no significant inhibition of the GABA-dependent activity. These results indicate that the basal route for 36Cl- entry into cerebral neurons involves electroneutral processes while the GABA-dependent influx occurs via specific ligand-gated Cl-channels.