The activation properties of GABAA receptors containing α4β2γ2 and α4β2δ subunits were examined in the presence of GABA or pentobarbital. The receptors were expressed transiently in HEK 293 cells, and the electrophysiological experiments were carried out using cell-attached single-channel patch clamp or whole-cell macroscopic recordings. The data show that GABA is a stronger activator of α4β2γ2 receptors than α 4β2δ receptors. Single-channel clusters were recorded from α4β2γ2 receptors in the presence of 10-5000 μM GABA. The maximal intracluster open probability was 0.35, with a half-maximal response elicited by 32 μM GABA. Simultaneous kinetic analysis of single-channel currents obtained at various GABA concentrations yields a channel opening rate constant of 250 s-1, and a KD of 20 μM. In contrast, only isolated openings were observed in the presence of GABA for the α4β2δ receptor. Pentobarbital was a strong activator of both α4β2γ2 and α4β 2δ receptors. The maximal cluster open probability, recorded in the presence of 100 μM pentobarbital, was 0.7. At higher pentobarbital concentrations, the cluster open probability was reduced, probably due to channel block. The results from single-channel experiments were confirmed by macroscopic recordings from HEK cells in the presence of GABA or pentobarbital.