Gangliosides are surface membrane components that have been suggested to play a significant role in the regulation of many cellular events including neuronal differentiation, growth and regeneration. We have chosen PC12 cell as a model system to study the influence of exogenously added glycosphyngolipids on in vitro differentiation and regeneration. A mixture of bovine brain gangliosides, (GM1 21%, GD1a 39.7%, GD1b 16% and GT 19%) or purified GM1 and GT respectively were added to culture media containing NGF on plating day and their effect was monitored on alternate days starting on day 5. The degree and rate of fiber outgrowth was significantly enhanced by media containing gangliosides at a concentration of 10(-6), 10(-7) M when serum was left out and 10(-3), 10(-4) M when serum was added to the culture medium. The stimulating ganglioside action required the presence of NGF to induce neurite outgrowth. However, binding studies indicated that exogenous gangliosides do not affect NGF binding to PC12 cells, therefore their stimulatory action may be separated from the interaction between NGF and its receptors. Subculturing of NGF-treated cells for 10 days demonstrated that gangliosides treatment also enhanced the NGF stimulated regeneration of neurites. Gangliosides may be incorporated at the level of cell surface, thereby affecting and facilitating membrane phenomena involved in neurite outgrowth.