The mechanisms governing the assembly of alpha-, beta- and gamma-subunits to form GABAA-receptors are poorly understood. Here, we report that the alpha-subunit is essential for receptor assembly. In mice homozygous for a deletion on chromosome 7 spanning the alpha 5- and gamma 3-subunit genes, zolpidem-insensitive benzodiazepine binding sites, corresponding to GABAA-receptors containing the alpha 5-subunit, were absent in the hippocampus. This loss of alpha 5-GABAA-receptor binding was also apparent as a 21% decrease in the total number of benzodiazepine binding sites in the hippocampus. In addition, immunoreactivity for the beta 2,3- and gamma 2-subunit was decreased exclusively in neurons which normally express the alpha 5-subunit, such as olfactory bulb granule cells and hippocampal pyramidal cells. In other brain regions of the mutants, the beta 2,3- and gamma 2-subunit staining was unaffected. Controls included two lines of mice homozygous for a shorter chromosomal deletion, that either included or excluded the gamma 3-subunit gene. These two lines were indistinguishable with regard to numbers of benzodiazepine binding sites and levels alpha 5-, beta 2,3- and gamma 2-subunit immunoreactivity, indicating that the lack of gamma 3-subunit gene did not contribute to the observed deficit in receptor formation. These results demonstrate that the absence of the alpha 5-subunit gene prevents the formation of the entire respective receptor complex in adult mouse brain. Thus, the alpha-subunit, unlike the gamma 2-subunit, might play a major role in the assembly or targeting of GABAA-receptor complexes.