Most general anesthetics produce two distinct actions at GABA(A) receptors. Thus, these drugs augment GABA-gated chloride currents (referred to as an indirect action) and, at higher concentrations, elicit chloride currents in the absence of GABA (referred to as a direct action). Because a beta subunit appears to be required for the direct action of intravenous anesthetics in recombinant GABA(A) receptors, site-directed mutagenesis of the beta3 subunit was performed to identify amino acid residues that are critical for this action. In HEK293 cells expressing a prototypical GABA(A) receptor composed of alpha1beta3gamma2 subunits, mutation of amino acid 290 from Asn to Ser dramatically reduced both etomidate-induced chloride currents and its ability to stimulate [3H]flunitrazepam binding. By contrast, the ability of etomidate to augment GABA-gated chloride currents and GABA-enhanced [3H]flunitrazepam binding was retained. The demonstration that the direct, but not the indirect, actions of etomidate are dependent on beta3(Asn290) indicates that the dual actions of this intravenous anesthetic at GABA(A) receptors are mediated via distinct loci.