Steroid hormone action involves binding to cognate intracellular receptors that, in turn, bind to respective response elements and thus modulate gene expression. The present study shows that the gonadal steroids, 17beta-estradiol and progesterone, may also act as functional antagonists at the 5-hydroxytryptamine type 3 (5-HT3) receptor in whole-cell voltage-clamp recordings of HEK 293 cells stably expressing the 5-HT3 receptor. Functional antagonistic properties at this ligand-gated ion channel could also be shown for 17alpha-estradiol, 17alpha-ethinyl-17beta-estradiol, mestranol, R 5020, testosterone, and allopregnanolone but not for pregnenolone sulfate and cholesterol. An antagonism at the 5-HT3 receptor could further be observed with the aromatic alcohol 4-dodecylphenol but not with phenol or ethanol. Thus, the modulation of 5-HT3 receptor function by steroids or alcohols is dependent on their respective molecule structure. The antagonistic action of steroids at the 5-HT3 receptor is not mediated via the serotonin binding site because the steroids did not alter the binding affinity of [3H]GR65630 to the 5-HT3 receptor, and kinetic experiments revealed a quite different response pattern to 17beta-estradiol when compared with the competitive antagonist metoclopramide. BSA-conjugated gonadal steroids labeled with fluorescein isothiocyanate bound to membranes of HEK 293 cells expressing the 5-HT3 receptor in contrast to native HEK 293 cells. However, there was no dose-dependent displacement of the binding of gonadal steroids to membranes of cells expressing the 5-HT3 receptor in binding experiments or fluorescence studies. Thus, gonadal steroids probably interact allosterically with the 5-HT3 receptor at the receptor-membrane interface. The functional antagonism of gonadal steroids at the 5-HT3 receptor may play a role for the development and course of nausea during pregnancy and of psychiatric disorders.