Mouse neuroblastoma N18 cells contain a homologous series of gangliosides (GM3, GM2, GM1, and GD1a) which constitute a biosynthetic pathway. When added to the culture medium, tritium-labeled palmitate, galactose, and N-acetylmannosamine were incorporated into these gangliosides. Incorporation of [3H]galactose into all four gangliosides was detected by 5 min and continued at essentially linear rates for several hours. When the cells were treated with Vibrio cholerae neuraminidase, the amounts of GM3 and GD1a were reduced from 72% to 85%; there was a severalfold increase in GM1 and no change in GM2. In spite of these large alterations in cellular ganglioside composition, there was no change in the rate of [3H]galactose incorporation into the gangliosides. A large proportion of GM3 and GD1a also was accessible to neuraminidase in neuroblastoma NB41A, Friend erythroleukemic, and rat glioma C6 cells. N18, NB41A, and Friend cells bound large amounts of 125I-labeled cholera toxin with high affinity. At saturation, the ratio of GM1 content to toxin bound for the three cell lines was between 5.5 and 7. When treated with neuraminidase, the cells bound more toxin in correspondence to the increase in GM1 content. As each toxin molecule has five binding sites, these results suggest that most of the GM1 in these cells is on the surface. Our results indicate that the sequential glycosylation of one ganglioside to form the next higher homologue involves a very small pool of intermediates and that the bulk of the gangliosides are on the cell surface.