Ganglioside composition was examined in an experimental mouse brain tumor growing as a solid tumor in vivo and as a cultured cell line in vitro. Gangliosides were also studied in the solid tumor rederived from the cultured tumor cell line. Although GM3-NeuAc was the major ganglioside in both the solid tumor and cultured tumor cells, several gangliosides expressed in the solid tumors (e.g., GM2-NeuGc, GM1, and GM1b) were not expressed in the cultured tumor cells. These gangliosides, however, are major components of mouse macrophages. Furthermore, significant amounts of gangliosides containing N-glycolylneuraminic acid (NeuGc) were found in the solid tumor growing in vivo, but only trace amounts were present in the cultured tumor cells. NeuGc is a common ganglioside sialic acid in mouse nonneural cells, whereas N-acetylneuraminic (NeuAc) is the predominant sialic acid in mouse brain. The trace amounts of NeuGc in the cultured cells are attributed to contamination from the fetal bovine serum. Radiolabeling of the cultured tumor cell gangliosides with [14C]galactose revealed that GM3-NeuAc was the only ganglioside synthesized by the tumor cells. The results suggest that nontumor-infiltrating cells, e.g., macrophages, lymphocytes, and endothelial cells, may contribute significantly to the total ganglioside composition of solid tumors growing in vivo.