Benzodiazepines (BZs) exert their therapeutic effects in the mammalian central nervous system at least in part by modulating the activation of gamma-aminobutyric acid (GABA)-activated chloride channels. To gain further insight into the mechanism of action of BZs on GABA receptors, we have been investigating structural determinants required for the actions of the BZ diazepam (dzp) on recombinant alpha1 beta2 gamma2 GABA(A) receptors. Site-directed mutagenesis was used to introduce point mutations into the alpha1 and gamma2 GABA(A) receptor subunits. Wild-type and mutant GABA(A) receptors were then expressed in Xenopus laevis oocytes or human embryonic kidney 293 (HEK 293) cells and studied using two-electrode voltage-clamp and ligand-binding techniques. With this approach, we identified two tyrosine residues on the alpha1 subunit (Tyr159 and Tyr209) that when mutated to serine, dramatically impaired modulation by dzp. The Y209S substitution resulted in a >7-fold increase in the EC50 for dzp, and the Y159S substitution nearly abolished dzp-mediated potentiation. Both of these mutations abolished binding of the high affinity BZ receptor antagonist [3H]Ro 15-1788 to GABA(A) receptors expressed in HEK 293 cells. These tyrosine residues correspond to two tyrosines of the beta2 subunit (Tyr157 and Tyr205) previously postulated to form part of the GABA-binding site. Mutation of the corresponding tyrosine residues on the gamma2 subunit produced only a slight increase in the EC50 for dzp (approximately 2-fold) with no significant effect on the binding affinity of [3H]Ro 15-1788. These data suggest that Tyr159 and Tyr209 of the alpha1 subunit may be components of the BZ-binding site on alpha1 beta2 gamma2 GABA(A) receptors.