Recombinant GABA(A) receptors, expressed from alpha-, beta-, and gamma2-subunits, are diazepam-insensitive when the alpha-subunit is either alpha4 or alpha6. In situ, diazepam-insensitive receptors containing the alpha6-subunit are almost exclusively expressed in the granule cell layer of the cerebellum. However, diazepam-insensitive receptors are also expressed in forebrain areas. Here, we report on the presence of diazepam-insensitive GABA(A) receptors in various brain areas containing the alpha4-subunit. GABA(A) receptors immunoprecipitated with a newly developed alpha4-subunit-specific antiserum displayed a drug binding profile that was indistinguishable from those of alpha4beta2gamma2-recombinant receptors and diazepam-insensitive [3H]Ro 15-4513 binding sites in rat brain membranes. In addition, alpha4-subunit containing receptors and forebrain diazepam-insensitive receptors are present at comparably low abundance in rat brain and exhibit virtually identical patterns of distribution. Analysis of the subunit architecture of alpha4-subunit containing receptors revealed that the alpha4-subunit contributes to several receptor subtypes. Depending on the brain region, the alpha4-subunit can be coassembled with a second type of alpha-subunit variant being alpha1, alpha2, or alpha3. The data demonstrate that native receptors containing the alpha4-subunit are structurally heterogeneous, expressed at very low abundance in the brain, and display the drug binding profile of diazepam-insensitive [3H]Ro 15-4513 binding sites. Pharmacologically, these receptors may contribute to the actions of nonclassical ligands such as Ro 15-4513 and bretazenil.