γ-Aminobutyric acid (GABAa) receptor β3 subunits were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamp. Injected oocytes exhibited an increased resting membrane conductance and more depolarized membrane potentials compared to uninjected control cells. Oocytes expressing β3 subunits were insensitive to GABA and muscimol, but pentobarbitone increased the membrane conductance in a concentration-dependent manner. The membrane current response to pentobarbitone reversed at the Cl−equilibrium potential and at relatively high concentrations (>500 μM), a rebound CI− current was induced following the removal of pentobarbitone. In transfected human embryonic kidney (HEK) cells, the rebound current amplitude was reduced by desensitizing the β3 receptor with increased durations of ligand application. Both picrotoxin (0.5 nM to 10 μM) and Zn2+ (10 nM to 100 μM) reduced the resting membrane conductance for β3 cDNA-injected oocytes. These oocytes were insensitive to flurazepam (5 μM) and alphaxalone (10 μM), but responded with increased membrane conductance to propofol (10 μM) and pregnanolone (50 nM to 5 μM). The antagonists, bicuculline (10 μM) and strychnine (50 nM to 100 μM), also induced conductance increases in a concentration dependent manner; however, glycine (1 mM) was inactive. It was concluded that β3 subunits form spontaneously opening ion channels that can be up-regulated by some allosteric modulators, principally by pentobarbitone and propofol and, surprisingly, by bicuculline and strychnine, whilst picrotoxin and Zn2+ acted as antagonists. Computer modelling of some kinetic schemes was used to describe the rebound current observed in transfected HEK cells. This indicated that pentobarbitone, after modulation of the conductance, is potentially capable of further binding to the β3 receptor complex ‘driving’ the receptor into one or more desensitized states. This phenomenon may be of some importance for native neuronal GABAA receptors, where pentobarbitone can also evoke rebound current activation.