Ethanol enhancement of 5-hydroxytryptamine (5-HT)(3A) receptor-mediated responses may have important consequences in the intoxicating and addictive properties of ethanol. Although the exact mechanism is unknown, ethanol-mediated enhancement of 5-HT(3) receptor current has been proposed to occur due to stabilization of the open-channel state. It has not been possible to directly measure the open state of the channel due to the extremely low single-channel conductance of 5-HT(3A) channels. Recently, three arginine residues within the large intracellular loop of the 5-HT(3A) subunit were substituted by their equivalent residues (glutamine, aspartate, and alanine) of the 5-HT(3B) subunit to produce a 5-HT(3A)(QDA) subunit that forms functional homomeric channels exhibiting a measurable single-channel conductance. Using whole-cell rapid-agonist application techniques and the cell-attached single-channel recording configuration, we examined human 5-HT(3A)(QDA) receptors expressed in human embryonic kidney 293 cells. The agonist sensitivity, macroscopic kinetics, and modulation by ethanol were similar between mutant and wild-type channels, suggesting the substitutions had not altered these channel structure-function properties. The open time histogram for single-channel events mediated by 5-HT(3A)(QDA) receptors in the presence of maximal 5-HT was best fit by three exponentials, but in the presence of ethanol a fourth open state was evident. In summary, the QDA substitution greatly enhanced single-channel conductance with little effect on 5-HT(3A) channel's kinetic properties and ethanol enhances agonist action on 5-HT(3A) receptors by inducing a new, long-lived open-channel state. Furthermore, the 5-HT(3A)(QDA) receptor appears to be suitable for pharmacological studies of 5-HT(3A) receptor modulation at a single-channel level.