1. We transiently introduced the human GABAA receptor ε subunit cDNA into a human embryonic kidney (HEK) cell line stably expressing α1β3γ2 receptors (WSS-1 cells) to establish whether the subunit competes with the γ2 subunit for assembly into receptors. GABA-evoked currents were recorded using the patch-clamp technique from cells transfected with cDNA encoding green fluorescent protein (GFP) alone or in combination with the ε subunit cDNA. 2. The ε subunit did not change the potency of GABA: the GABA EC50 was 34 ± 6 μM in control WSS-1 cells and 37 ± 6 μM in cells expressing the ε subunit. The introduction of the ε subunit reduced the peak current amplitude activated by GABA (1 mM) from 1.8 ± 0.2 nA in control cells to 0.9 ± 0.2 nA in cells expressing the ε subunit (P < 0.05). 3. The ε subunit caused the appearance of leak currents recorded in the absence of GABA. Outside-out patches excised from ε subunit-containing WSS-1 cells exhibited spontaneously opening GABAA channels not seen in patches excised from control GFP-expressing WSS-1 cells. Introduction of the ε subunit did not alter the GABA-evoked single-channel cord conductance. 4. The anaesthetic 2,6-diisopropylphenol (propofol, 3 μM) and the benzodiazepine flunitrazepam (1 μM) potentiated GABA-evoked currents recorded from control cells labelled with GFP. The ε subunit reduced potentiation by both agents 48-96 h after transfection. 5. The introduction of the ε subunit had no effect on the ability of propofol (3-30 μM) relative to GABA (1 mM) to activate GABAA receptors in WSS-1 cells. High concentrations of propofol (≥ 100 μM) produced a more marked desensitization of GABAA receptor activity in WSS-1 cells transfected with cDNA for the ε subunit than in control cells. 6. There was no difference in the potency of Zn2+ as an inhibitor of currents recorded from control cells (IC50 = 165 ± 34 μM) or cells expressing the ε subunit (IC50 = 179 ± 11 μM). 7. GABA-activated currents recorded both from control cells and cells expressing the ε subunit reversed in sign at the CI- equilibrium potential and exhibited outward rectification. 8. The introduction of the ε subunit changes the functional properties of GABAA receptors in WSS-1 cells. The resulting receptors have a unique combination of properties indicative of the co-assembly of α, β, γ and ε subunits.