1. To determine their contributions to GABAA receptor (GABAR) channel properties, rat gamma 2L and delta subunits were acutely co-expressed with alpha 1 and beta 3 subtypes in mouse L929 fibroblasts to produce alpha 1 beta 3, alpha 1 beta 3 delta or alpha 1 beta 3 gamma 2L GABAR isoforms. 2. With whole-cell recording, the alpha 1 beta 3 isoform had relatively high sensitivity to GABA (EC50 2.1 microM) and low maximum current amplitude. The alpha 1 beta 1 delta isoform had similar sensitivity to GABA (EC50 2.8 microM) and low current amplitude. The alpha 1 beta 3 gamma 2L isoform had lower sensitivity to GABA (EC50 11.6 microM) and higher maximum current amplitude. 3. The single channel conductance of alpha 1 beta 3 channels was low (13 pS) compared with that of alpha 1 beta 3 delta and alpha 1 beta 3 gamma 2L channels (27 pS). 4. The single channel kinetic properties of the channels also differed. The alpha 1 beta 3 gamma 2L channel exhibited three open states, while the alpha 1 beta 3 and alpha 1 beta 3 delta channels exhibited only two open states with mean dwell times similar to those of the two shorter open states of the alpha 1 beta 3 gamma 2L channel. All three channels exhibited at least five closed states. Bursts of alpha 1 beta 3 delta channels consisted primarily of only one or two openings, while those of alpha 1 beta 3 channels contained multiple openings. alpha 1 beta 3 gamma 2L channels exhibited burst kinetics typical for native GABARs with several long openings per burst. 5. These results show that the alpha 1 beta 3 heterodimer formed a GABA-sensitive channel with complex gating kinetics. Addition of a delta subunit to form the alpha 1 beta 3 delta heterotrimer altered the single channel conductance and the kinetic properties of the closed components, but did not affect the GABA sensitivity of the receptor nor the open state kinetics. In contrast, addition of a gamma subunit (gamma 2L subtype) to produce the alpha 1 beta 3 gamma 2L heterotrimer affected the GABA sensitivity, channel conductance and kinetic properties of both open and closed states.