Developmental changes in ganglioside levels and patterns were investigated in neuronal cells dissociated from 17-day-old fetal rat hemispheres for up to 7 days of culture. Increases in ganglioside contents and the onset of GM3 synthesis, which is associated with proliferation of glial cells, were observed as the neuronal network was established in cell cultures. The distribution of gangliosides in developing neurons was monitored by the indirect immunofluorescent technique using three anti-ganglioside antibodies. Anti-GM1 antibody showed immunofluorescence only on the cell soma 1 and 3 days after plating and additional binding between cell aggregates by 7 days in culture. GD3 ganglioside, the predominant species in embryonic neurons, was not detected on the neuronal cell surface, whereas the number of positively stained non-neuronal cells was increased at 7 days. Monoclonal A2B5 antibody suggested that polysialogangliosides play a role in neuronal network formation. In 1-day-old culture, however, all antibodies bound poorly to cell surface antigens and strongly to cells, the membranes of which were permeabilized with acetone. These results suggest that a substantial amount of gangliosides are retained, transformed within the cell to more complex gangliosides, and translocated to the cell surface following neurite outgrowth and morphological changes.