Human melanoma cells express relatively large amounts of the disialogangliosides GD3 and GD2 on their surface whereas neuroblastoma cells express GD2 as a major ganglioside. Monoclonal antibodies (Mabs) directed specifically to the carbohydrate moiety of GD3 and GD2 inhibit melanoma and neuroblastoma cell attachment to various substrate adhesive proteins, e.g. collagen, vitronectin, laminin, fibronectin, and a heptapeptide, glycyl-L-arginyl-glycyl-L-aspartyl-L-seryl-L-prolyl-L-cysteine, which constitutes the cell attachment site of fibronectin. Cells that are preattached to a fibronectin substrate can also be induced to detach and round up in the presence of purified anti-ganglioside Mab. Moreover, when melanoma cells that contain both GD2 and GD3 are incubated with Mabs directed to both of these molecules an additive inhibition is observed. The specificity of this inhibition is demonstrated since Mabs of various isotypes directed to either protein or carbohydrate epitopes on a number of other major melanoma or neuroblastoma cell surface antigens have no effect on cell attachment. A study of the kinetics involved in this inhibition indicates that significant effects occur during the first 5 min of cell attachment, suggesting an important role for GD2 and GD3 in the initial events of cell-substrate interactions. The role of gangliosides in cell attachment apparently does not directly involve a strong interaction with fibronectin since we could not observe any binding of radiolabeled fibronectin or fragments of the molecule known to contain the cell attachment site to melanoma gangliosides separated on thin-layer chromatograms. An alternative explanation would be that gangliosides may play a role in the electrostatic requirements for cell-substrate interactions. In this regard, controlled periodate oxidation of terminal, unsubstituted sialic acid residues on the cell surface not only specifically destroys the antigenic epitopes on GD2 and GD3 recognized by specific Mabs but also inhibits melanoma cell and neuroblastoma cell attachment. In fact, the periodate-induced ganglioside oxidation and the inhibition of cell attachment are equally dose dependent. These data suggest that cell-substratum interactions may depend in part on the electrostatic environment provided by terminal sialic acid residues of cell surface gangliosides and possibly other anionic glycoconjugates.