Gangliosides were employed as early differentiation markers to investigate how phenotypic diversity is generated in the vertebrate neutral crest cell population. Chromatographic analysis of metabolically labeled glycolipids from neural crest derivatives revealed that glia cell precursors synthesize a characteristic subset of the ganglioside types produced by dorsal root ganglion neurons. The ganglioside synthesis pattern of neural crest cultures is similar to that of glial precursors, but gangliosides characteristic of neurons are also detectable. To determine whether neuronal gangliosides are expressed by every cell in neural crest cultures, or by discrete cell subpopulations, crest cultures were stained immunocytochemically with monoclonal antibody A2B5 which recognizes a neuron-specific ganglioside in the GQ fraction. A2B5 was found to bind to about 1% of migratory stage neural crest cells isolated from neural tube explants after 1 day in culture. These A2B5+ cells were postmitotic and exhibited a uni- or bipolar neuronal morphology. A second, larger (10-20%) population of A2B5+ cells appears after culturing cells from 1-day crest cell clusters an additional 1-2 days. These cells initially have the typical small, stellate morphology of crest cells but later extend one or more processes. [3H]Thymidine incorporation and cell counting studies show that the precursors to these cells had divided at least once in culture before becoming postmitotic and expressing A2B5 immunoreactivity. The second A2B5+ population does not appear in secondary cultures of crest cell clusters isolated from 2-day-old explants of neural tubes. Another monoclonal antibody, R24, which recognizes ganglioside GD3, binds to subpopulations of both neurons and nonneurons in sensory ganglion cultures. In neural crest cultures R24 binds to a large subpopulation of cells, but not to A2B5+ ones. The significance of this immunostaining pattern is not yet understood. The early appearance of subpopulations, and the presence of heterogeneity in neural crest cultured under a variety of conditions suggest that intrinsic cellular mechanisms might generate subpopulations within the neural crest upon which environmental factors act.