Abstract. The patch-clamp technique was used on excised (outside-out) patches to characterize h5-HT3A receptors stably transfected in HEK 293 cells and to compare the effects of the barbiturate anaesthetics methohexital and pentobarbital on this ligand-gated cation channel. At negative membrane potentials 5-HT induced inward currents in a concentration-dependent manner (EC50=8.6M, Hill coefficient =1.5). The mean peak current induced by 30M 5-HT was 110pA at 100mV. The 5-HT3A receptor antagonist ondansetron (0.3nM) reversibly inhibited the 5-HT (30M) signal by 70% and at 3nM it abolished the response. Methohexital and pentobarbital inhibited 5-HT-induced (30M) currents in a concentration-dependent manner. The maximal inhibition with a given methohexital or pentobarbital concentration was reached when the respective drug was applied 45s prior to and during the 2-s 5-HT pulse (IC50 values=95M and 127M, Hill coefficient =1.0 and 1.6, respectively). Although the barbiturates were, thus, equipotent, their effects differed substantially with respect to the dependence on the time schedule of application to the patches: the potency of methohexital was virtually maximal when the drug was applied exclusively 45s before the agonist pulse, but its inhibitory potency decreased considerably when it was exclusively applied during the 2-s 5-HT pulse (IC50=380M). Conversely, pentobarbital was almost maximally potent in inhibiting the 5-HT signal when it was exclusively coapplied with this agonist, but its inhibitory potency was considerably lower (IC50 ~500M) when applied exclusively 45s before 5-HT. Another difference between both barbiturates involves the rate of inactivation of 5-HT3 receptor-mediated currents: whereas high concentrations of methohexital (300M) were necessary to induce moderate ( twofold) acceleration of this parameter, pentobarbital produced such an effect at all concentrations and the extent of acceleration increased with increasing concentration (1.5- to fivefold). In conclusion, two barbiturates, chemically closely related but of different lipophilicity, clearly differ with respect to the kinetics of their effect on 5-HT3 receptor channels; one possible explanation involves drug access to an amphipathic site of action via both an aqueous and a hydrophobic pathway. Pentobarbital, in contrast to methohexital, inhibits h5-HT3A receptor-mediated currents at anaesthetic concentrations (~90M).