GABA(A) receptor subtypes comprising the alpha1 and alpha3 subunits change with development and have a specific anatomical localization in the adult brain. These receptor subtypes have been previously demonstrated to greatly differ in deactivation kinetics but the underlying gating mechanisms have not been fully elucidated. Therefore, we expressed rat alpha1beta2gamma2 and alpha3beta2gamma2 receptors in human embryonic kidney 293 cells and recorded current responses to ultrafast GABA applications at macroscopic and single-channel levels. We found that the slow deactivation of alpha3beta2gamma2-mediated currents is associated with a relatively small rate and extent of apparent desensitization. In contrast, responses mediated by alpha1beta2gamma2 receptors had faster deactivation and stronger desensitization. Alpha3beta2gamma2 receptors had faster recovery in the paired-pulse agonist applications than alpha1beta2gamma2 channels. The onset of currents mediated by alpha3beta2gamma2 receptors was slower than that of alpha1beta2gamma2 for a wide range of GABA concentrations. Single-channel analysis did not reveal differences in the opening/closing kinetics of alpha1beta2gamma2 and alpha3beta2gamma2 channels but burst durations were longer in alpha3beta2gamma2 receptors. Simulation with a previously reported kinetic model was used to explore the differences in respective rate constants. Reproduction of major kinetic differences required a smaller desensitization rate as well as smaller binding and unbinding rates in alpha3beta2gamma2 compared with alpha1beta2gamma2 receptors. Our work describes the mechanisms underlying the kinetic differences between two major GABA(A) receptor subtypes and provides a framework to interpret data from native GABA receptors.