The physiological and pharmacological properties of GABAA receptors have been studied extensively after the expression of subunits in non-neural cells. Many of these studies have used the human embryonic kidney cell line HEK 293. We examined the properties of subunits that result in the expression of low levels of functional receptors and found that the properties of the gamma-aminobutyric acid (GABA)-elicited responses in transfected HEK 293 cells differ from expectations based on previous work and are consistent with the idea that the expressed receptors do not necessarily contain the expected subunits. In particular, expression of a mutated beta 2 subunit [beta 2(Y205S)] in combination with alpha 1 and gamma 2L results in cells that have large responses to pentobarbital (as expected) but also show appreciable responses to GABA (contrary to expectation). Furthermore, transfection of HEK 293 cells with alpha 1 plus gamma 2L subunits results in responses to GABA that are potentiated by the drug loreclezole, suggesting that a subunit resembling the beta 2 or beta 3 subunit had assembled with the alpha 1 gamma 2L subunits. In addition, some nontransfected HEK 293 cells respond to applications of GABA, and transfection of cells with alpha 1, beta 1, or gamma 2L subunits alone can result in the expression of GABA-elicited currents. In comparison, when QT6 quail fibroblasts are used as the expression system, no responses were seen in untransfected cells or in cells transfected with alpha 1, beta 1, or gamma 2L subunits alone or alpha 1 gamma 2L subunits. Furthermore, no response to GABA was seen in QT6 cells transfected with alpha 1 beta 2(Y205S) gamma 2L subunits, although cells gave strong responses to pentobarbital. These observations indicate that caution must be taken in interpreting the results of studies of the properties of GABAA receptors expressed in HEK 293 cells if the exogenous subunits result in the expression of low levels of functional GABAA receptors.