We studied the effects of extracellular pH (pH(o)) on γ-aminobutyric acid (GABA)mediated C1- current in rat hypothalamic neurons and recombinant type-A GABA (GABA(A)) receptors stably expressed in human embryonic kidney cells (HEK 293), using whole cell and outside-out patch-clamp recordings. In α3β2γ2s receptors, acidic pH decreased, whereas alkaline pH increased the response to GABA in a reversible and concentration-dependent manner. Acidification shifted the GABA concentration-response curve to the right, significantly increasing the EC50 for GABA without appreciably changing the slope or maximal current induced by GABA. We obtained similar effects of pH in α1β3γ2 receptors and in GABA-activated currents recorded from thin hypothalamic brain slices. In outside-out patches recorded from α3β2γ2 recombinant receptors, membrane patches were exposed to 5 μM GABA at control (7.3), acidic (6.4), or alkaline (8.4) pH. GABA activated main and subconductance states of 24 and 16 pS, respectively, in α3β2γ2 receptors. Alkaline pH(o) increased channel opening frequency and decreased the duration of the long closed state, resulting in an increase in open probability (from 0.0801 ± 0.015 in pH 7.3 to 0.138 ± 0.02 in pH 8.4). Exposure of the channels to acidic pH(o) had the opposite effects on open probability (decreased to 0.006 ± 0.0001). Taken together, our results indicate that the function of GABA(A) receptors is modulated by extracellular pH. The proton effect is similar in recombinant and native receptors and is dependent on GABA concentration. In addition, the effect appears to be independent of the α-subunit isoform, and is due to the ability of H+ to alter the frequency of channel opening. Our findings indicate that GABAergic signaling in the CNS may be significantly altered during conditions that increase or decrease pH.