Gangliosides of the plasma membrane are important modulators of cellular functions. Recent reports have shown their enrichment in glycosphingolipid-containing membrane microdomains, called glycosphingolipid-signaling domain or rafts, which can be isolated due to their insolubility in Triton X-100 and flotation through a sucrose gradient. In previous work on neuroblastoma cells we had found that a ganglioside-specific sialidase activity of the plasma membrane controlled proliferation and differentiation through selective ganglioside desialylation. Assuming the ganglioside sialidase to be close to its substrates in the membrane, we investigated its association with detergent-insoluble microdomains in the neuroblastoma cell line SK-N-MC. The results show that the ganglioside sialidase codistributes with the raft markers ganglioside GM1, flotillin, src family kinases, and glycosylphosphatidylinositol-anchored proteins in a fraction containing about 2% of cellular protein. The association of the ganglioside sialidase with glycosphingolipid-enriched membrane fractions therefore is in support of a role of this glycosidase in ganglioside-dependent signaling processes.