Gangliosides may act as the cell surface receptor for fibronectin, as previously suggested by the data of Kleinman et al. Gangliosides inhibited the spreading of both CHO and BHK cells on fibronectin-coated substrates. Inhibition (50%) of cell spreading was produced by 1.0 and 3.5 .times. 10-5 M di- and trisialogangliosides, respectively, although monosialogangliosides were less effective inhibitors. The inhibition was apparently due to an interaction of gangliosides with fibronectin and not due to a direct effect of gangliosides on the cells. Anti-fibronectin antibodies, 125I-labeled protein A, and gangliosides adsorbed to polystyrene tubes, provided direct evidence that fibronectin will bind to gangliosides. The interaction is apparently of low affinity compared with binding of cholera toxin to gangliosides. The ability of BALB/c 3T3 cells to spread on fibronectin was compared with that on substrates coated with a ganglioside-specific ligand, cholera toxin B-subunit. Cells plated onto fibronectin-coated substrates rapidly (within 60 min) adopted a well spread morphology, whereas spreading on substrates coated with the toxin B-subunit was less extensive even after 2 h. The organization of F-actin within cells spread on the 2 types of substrate was also quite different. The interaction of cells with fibronectin may well be influenced by membrane-bound gangliosides. Binding of fibronectin to such gangliosides probably cannot lead to the cytoskeletal reorganization which is characteristic of cells spread on fibronectin.