Etomidate and propofol have clearly distinguishable effects on the central nervous system. However, studies in knock-in mice provided evidence that these agents produce anesthesia via largely overlapping molecular targets, namely GABA(A) receptors containing beta3 subunits. Here the authors address the question as to whether etomidate and propofol are targeting different subpopulations of beta3 subunit containing GABA(A) receptors. The effects of etomidate and propofol (0.5 muM and 1.0 muM) on spontaneous activity of neocortical neurons were investigated in organotypic slice cultures from wild-type and beta3(N265M) knock-in mice. Firing patterns were characterized by mean burst length and number of action potentials per burst. Additionally, etomidate and propofol actions on GABA(A) receptor-mediated currents were investigated by whole-cell voltage clamp recordings. On the network level, the duration of spontaneously occurring bursts of action potentials was decreased by etomidate but increased by propofol in the wild-type. The effects of etomidate were abolished in beta3(N265M) mutant slices while those of propofol were qualitatively inverted. On the receptor level, GABA(A) receptor-mediated inhibition of cortical neurons was modulated by etomidate and propofol in different ways. Again, drug-specific actions of etomidate and propofol were largely attenuated by the beta3(N265M) mutation. Etomidate and propofol alter the firing patterns and GABA(A) receptor-mediated inhibition of neocortical neurons in different ways. This suggests that etomidate and propofol act via non-uniform molecular targets. Because the major effects induced by these anesthetics were attenuated by the beta3(N265M) mutation, different subpopulations of beta3-containing GABA(A) receptors are likely to be involved.