Up-regulation of the GABAA receptor alpha4 subunit subtype has been consistently shown in multiple animal models of chronic epilepsy. This isoform is expressed in both thalamus and hippocampus and is likely to play a significant role in regulating corticothalamic and hippocampal rhythms. However, little is known about its physiological properties, thus limiting understanding of the role of alpha4 subtype-containing GABAA receptors in normal and abnormal physiology. We used rapid GABA application to recombinant GABAA receptors expressed in HEK293T cells to compare the macroscopic kinetic properties of alpha4beta3gamma2L receptors to those of the more widely distributed alpha1beta3gamma2L receptors. These receptor currents had similar peak current amplitudes and GABA EC50 values. However, alpha4beta3gamma2L currents activated more slowly when exposed to submaximal GABA concentrations, had more fast desensitization (tau = 15-100 ms), and had less residual current during long GABA applications. In addition, alpha4beta3gamma2L currents deactivated more slowly than alpha1beta3gamma2L currents. Peak currents evoked by repetitive, brief GABA applications were more strongly attenuated for alpha4beta3gamma2L currents than alpha1beta3gamma2L currents. Moreover, the time required to recover from desensitization was prolonged in alpha4beta3gamma2L currents compared to alpha1beta3gamma2L currents. We also found that exposure to prolonged low levels of GABA, similar to those that might be present in the extrasynaptic space, greatly suppressed the response of alpha4beta3gamma2L currents to higher concentrations of GABA, while alpha1beta3gamma2L currents were less affected by exposure to low levels of GABA. Taken together, these data suggest that alpha4beta3gamma2L receptors have unique kinetic properties that limit the range of GABA applications to which they can respond maximally. While similar to alpha1beta3gamma2L receptors in their ability to respond to brief and low frequency synaptic inputs, alpha4beta3gamma2L receptors are less efficacious when exposed to prolonged tonic GABA or during repetitive stimulation, as may occur during learning and seizures.