Anion-exchangers (AE) exchange intracellular bicarbonate for extracellular chloride and thus usually lower intracellular pHi. AE3, encoded by Slc4a3, is predominantly expressed in the heart and brain. Though the function of AE3 in vivo is largely unknown, the variant Ala867Asp has been associated with common subtypes of idiopathic generalized epilepsy. To explore, whether AE3 itself may be involved in the pathogenesis of seizures, we generated an AE3-knockout mouse model by targeted disruption of Slc4a3. AE3-knockout mice were apparently healthy and neither showed gross histological nor behavioral abnormalities. However, the seizure threshold of AE3-knockout mice exposed to bicuculline or pentylenetetrazole was dramatically reduced and seizure-induced mortality significantly increased compared to wild-type littermates. Nevertheless, no spontaneous seizures or spike wave complexes were found in electrocorticograms of wake adult AE3-knockout mice. In hippocampal CA3 neurons, disruption of AE3 reduced the ability to regulate alkalotic shifts of the intracellular pH. Moreover, the intraneuronal alkalosis upon withdrawal of extracellular chloride was abolished, indicating that AE3-knockout mouse pyramidal neurons were devoid of sodium-independent chloride-bicarbonate-exchange. Neuronal hyperexcitability was also confirmed by electrophysiological analysis of hippocampal slices of AE3-knockout mice. These findings strongly support the hypothesis