In vivo priming of lymphocytes by gangliosides was tested following active immunization of mice with the tetrasialoganglioside GQ1b coupled to a copolymer of L-lysine and DL-alanine. Lymphocytes derived from potential lymph nodes 17 days after immunization were triggered to incorporate tritiated thymidine after a 4-day incubation in polypropylene tubes precoated with GQ1b. Non-primed lymph node lymphocytes, primed lymphocytes incubated in non-coated tubes, and, more interestingly, primed lymphocytes incubated with the soluble ganglioside did not undergo mitogenic stimulation. The data suggest that the evaluation of the extent of in vivo priming of lymphocytes by gangliosides can be achieved only if these glycosphingolipids are bound to the substratum, whereas when applied to primed cells in soluble form they inhibit mitogenic activation.