AbstractGanglioside GM1 is the receptor for cholera toxin on cell surfaces, and the binding of cholera toxin to GM1 immobilized on microtitre plates has been reported previously by several authors as an assay for the toxin (GM1‐ELISA). This assay has been examined in detail. Results were independent of the adsorption solvent for GM1 (methanol or phosphate‐buffered saline), the pH of aqueous solvents (7.4–10.2) and the temperature (4–37 °C). High and near‐maximal rates of absorbance change in the assay were found for lower concentrations of GM1 (100 ng ml−1) and for shorter incubation times (a few hours) than reported in the literature. A method was devised to provide a semi‐quantitative estimate of the amount of GM1 bound to the plate; this was found to be in the low nanogram range. Binding of cholera toxin to the immobilized GM1 required ≥1.5 h for maximal assay results. The failure of free GM1 in solution to displace cholera toxin once bound to immobilized GM1 indicated that binding to immobilized GM1 is irreversible in the time frame of the experiment. Data from the literature support the very slow dissociation rates of the toxin–GM1 complex. Copyright © 2005 John Wiley & Sons, Ltd.