Juvenile myoclonic epilepsy (JME) belongs to the most common forms of hereditary epilepsy. The first mutation was recently detected in the a1-subunit (A322D) of the GABAA receptor. We now studied the functional consequences of this mutation in molecular detail using co-expression of a1-, a1-A322D-, b2- and [gamma]2-subunits in HEK293 cells. We investigated the specificity of the effects for GABA- versus Phenobarbital-induced gating on the A322D mutation. The a1-A322D mutation leads to a severe loss-of-function of the human GABAA receptor by reduced surface expression, reduced GABA-sensitivity, and accelerated deactivation. In contrast to GABA, Phenobarbital shows no reduced agonistic effect on the mutant receptor compared to the wild type. The next step, we investigated GABA and/or Phenobarbital and the antagonistic effect of the competitive blocker Bicuculline on GABAA receptor channels. The antagonistic effect was significantly reduced on the a1-A322D GABAA receptor using GABA as agonist but unchanged using Phenobarbital as agonist. This, further, supports the view of independent channel activation pathways elicited by GABA- or Barbiturate- binding.