A GABA(A) receptor delta subunit-deficient mouse line was created by homologous recombination in embryonic stem cells to investigate the role of the subunit in the brain GABA(A) receptors. High-affinity [(3)H]muscimol binding to GABA sites as studied by ligand autoradiography was reduced in various brain regions of delta(-/-) animals. [(3)H]Ro 15-4513 binding to benzodiazepine sites was increased in delta(-/-) animals, partly due to an increment of diazepam-insensitive receptors, indicating an augmented forebrain assembly of gamma 2 subunits with alpha 4 subunits. In the western blots of forebrain membranes of delta(-/-) animals, the level of gamma 2 subunit was increased and that of alpha 4 decreased, while the level of alpha1 subunits remained unchanged. In the delta(-/-) forebrains, the remaining alpha 4 subunits were associated more often with gamma 2 subunits, since there was an increase in the alpha 4 subunit level immunoprecipitated by the gamma 2 subunit antibody. The pharmacological properties of t-butylbicyclophosphoro[(35)S]thionate binding to the integral ion-channel sites were slightly altered in the forebrain and cerebellum, consistent with elevated levels of alpha 4 gamma 2 and alpha 6 gamma 2 subunit-containing receptors, respectively.The altered pharmacology of forebrain GABA(A) receptors and the decrease of the alpha 4 subunit level in delta subunit-deficient mice suggest that the delta subunit preferentially assembles with the alpha 4 subunit. The delta subunit seems to interfere with the co-assembly of alpha 4 and gamma 2 subunits and, therefore, in its absence, the gamma 2 subunit is recruited into a larger population of alpha 4 subunit-containing functional receptors. These results support the idea of subunit competition during the assembly of native GABA(A) receptors.