The interaction of cholera toxin and a number of toxin derivatives, containing different proportions of light and heavy toxin-composing subunits (L and H), with mouse lymphocytes was studied. Experiments with [(125)I]toxin showed that a single cell can rapidly, within minutes, bind up to 40,000 molecules of toxin, the association constant was estimated to 7 +/- 4 x 10(8) liters/mol, and binding was found to be very similar at 37 degrees C and 5 degrees C. Immunofluorescence studies revealed that the toxin attachment is located on the cell surface, and that purified L subunit but not H subunit binds to the cells. A natural cholera toxoid, built up by aggregated L subunits, showed almost identical binding properties as toxin to the cells. Pure G(M1) ganglioside, the proposed membrane receptor structure for toxin, prevented entirely the cellular binding of both toxin and toxoid. Cholera toxin in concentrations down to approximately 5 x 10(-11) mol/liter (corresponding to 10 bound molecules/cell) inhibited thymus cells from being stimulated to DNA synthesis by concanavalin A (con A), and spleen cells from such stimulation by phytohemagglutinin. The G(M1) ganglioside but not a series of other pure structurally related gangliosides and neutral glycosphingolipids neutralized this toxin activity. Toxin derivatives which, in similarity with toxin, possessed H as well as L subunits but in other proportions, were potent inhibitors of con A-induced thymocyte stimulation, whereas the natural toxoid (aggregated L subunits), purified toxin L subunit and purified toxin H subunit were up to 300-fold less active on a weight basis. The capacity of cholera proteins to inhibit con A-induced thymocyte stimulation correlated well with their activity in the rabbit intradermal toxicity assay. The inhibitory action of cholera toxin on con A-induced thymocyte stimulation did not depend on decreased cell viability from the toxin treatment, nor was it caused by a reaction between toxin and con A. [(125)I]con A bound equally well to the cells when toxin was present as when it was absent, which proves that the toxin did not compete for cellular con A receptors. Nor did the toxin seem to disturb the general mobility of membrane receptors or their ability to accumulate in caps. It is concluded that the L type of subunit confers rapid and firm binding of cholera toxin to lymphocyte membranes, probably to G(M1) ganglioside receptors. For biologic activity the additional presence of H subunit is important. One manifestation of toxin action on lymphocytes is inhibition of lectin-induced DNA synthesis; probably this effect relates to the ability of cholera toxin to raise the levels of intracellular cyclic 3'5'-adenosine monophosphate.