GABAA receptors are inhibitory transmitter-gated ion channels formed of various combinations of the 19 homologous subunits cloned to date. Changes in extracellular pH have been shown to directly activate the receptor or modulate its activity elicited by a chemical agonist, but the direction and magnitude of the effect depend on the subunit composition of the receptor. Here, we investigated the acute effect of protonation on the function of the human β3 homomeric GABAA receptor. We show that the reduction of the pH of the extracellular bath solution directly activates this receptor. H+ concentration-response data yielded a pEC50 of ∼6 (EC50 ∼ 1 μM). The probability of being in the active state ranged from 0.005 at pH 8.5 to 0.37 at saturating [H+]. The activating effect of H+ was rapidly reversible, and H+-activated receptors showed minimal desensitization. Mutation of the conserved histidine residue in the 17' position in the second membrane-spanning helix (H267) to alanine abolished H+-elicited activation, revealing inhibition of constitutive activity at low pH that, in its turn, was removed by additionally introducing the H107G mutation in the extracellular domain. We propose that the previously reported constitutive activity of the β3 receptor at physiological pH reflects H+-elicited activity, expressing the net effect of protonation of H267 and H107 residues.