1. We have studied the kinetic properties of channel gating of recombinant α1β2γ2L GABAA receptors transiently expressed in human embryonic kidney 293 cells, using the cell-attached, single-channel patch-clamp technique. The receptors were activated by GABA, β-alanine or piperidine-4-sulfonic acid (P4S), and the effects of pentobarbital (PB) on single-channel activity were examined. 2. At relatively high concentrations of agonist, single-channel activity occurred in well-defined clusters. In global terms, PB increased the mean open time for events in clusters, without changing the mean closed time. The addition of PB shifted the curve relating the probability of being open in a cluster (Po) to lower agonist concentrations, and that shift could be accounted for by the changes in mean open time. 3. The intracluster closed-time histograms contained four components. The durations and relative frequencies of these closed-dwell components were not affected by the presence of 40 μM PB, at any agonist concentration. The duration of one component was dependent upon the concentration of agonist used to activate the receptor. Accordingly, the inverse of the mean duration of this component will be called the effective opening rate. 4. The channel-opening rate constant (β) was determined from the value of the effective opening rate at a saturating agonist concentration. β was about 1900 s-1 when the receptors were activated by GABA, 1500 s-1 when activated by β-alanine, and too low to be determined when P4S was administered. In the presence of 40 μM PB, β was about 1500 s-1 when the receptors were activated by GABA, 1400 s-1 when activated by β-alanine, and 50 s-1 when activated by P4S. Hence, the potentiating effect of PB is not mediated by a change in β. The concentration of agonist producing a half-maximal effective opening rate also remained unaffected in the presence of PB, indicating that receptor affinity for agonists is not influenced by PB. 5. The distributions of the intracluster open durations elicited by GABA could be described by the sum of three exponentials, with mean durations of about 0.4, 2.4 and 6.3 ms. The duration and relative frequency of the components did not change with GABA concentration (20 μM to 1 mM). In the presence of 40 μM PB, however, the mean duration of the longest of the open times increased (mean durations of about 0.4, 2.0 and 13 ms). The intracluster open durations elicited by β-alanine could be described by the sum of two exponential components (1.1 and 3.5 ms). However, in the presence of 40 μM PB the open-time distribution contained three exponential components (0.2, 2 and 10 ms). Finally, openings elicited by P4S exhibited two components (0.3 and 0.9 ms). In the presence of 40 μM PB, three components could be distinguished (0.5, 2.5 and 13 ms). 6. These observations indicate that the potentiating effect of PB on GABA type A (GABAA) receptors reflects effects on the open state(s) of the receptors. In the case of receptors activated by GABA, the observations are consistent with the idea that the action is the result of PB stabilizing one of the open states. The actions on receptors activated by P4S or β-alanine are also broadly consistent with this idea. However, the changes in open-time distributions caused by PB appear to be more complex. Possible explanations of the effects of PB on gating by different agonists are considered.