By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 mu in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process procedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types.