Gangliosides shed by tumor cells are immunosuppressive molecules, but the mechanisms of shedding are poorly understood. We therefore conducted a comprehensive study of shedding to identify the natural forms of shed gangliosides. By chemical detection and mass spectrometric analysis of the gangliosides of YAC-1 murine lymphoma cells, we first confirmed that all major ganglioside species are released. Then, by the combination of metabolic and cell surface radiolabeling, we further demonstrated that gangliosides are released directly from the cell plasma membrane, i.e. by shedding. Ultracentrifugation separated the conditioned medium of metabolically radiolabeled cells cultured in either serum-free or serum-containing medium into: (1) a pellet of 100-200 nm membrane vesicles (visualized by electron microscopy) containing nearly one-third of total shed gangliosides; and (2) the supernatant, which contained soluble gangliosides (two-thirds of the total shed gangliosides). Although the ganglioside concentration in the conditioned medium (6-14x10-8 M) was above the critical micelle concentration of purified YAC-1 gangliosides (<1x10-8 M), by gel filtration >90% of the soluble gangliosides were found in monomeric form (MW <2 kDa) and only <10% in micelles (130 kDa). Ultrafiltration of fresh conditioned medium likewise showed the existence of monomers, and the findings were confirmed in human Daoy medulloblastoma and mouse MEB4 melanoma cells. Thus, in their natural states, shed tumor cell gangliosides exist in three forms: membrane vesicles, micelles, and monomers.