Despite recent advances in our understanding of neural stem cell (NSC) biology, the free-floating structures generated by these cells in vitro, the "neurospheres", have not been fully characterized. To fill this gap, we examined neurospheres and neurosphere-derived NSCs by confocal microscopy, electron microscopy (EM) and cytofluorimetry. Here, we show that neurospheres and neurosphere-forming cells are morphologically and functionally heterogeneous. Confocal microscopy reveals differences in cell size, viability, cytoplasmic content and in the presence and distribution of active mitochondria. By electron microscopy, neurospheres appear as complex structures in which biological events such as mitosis, apoptosis and even phagocytosis are influenced by NSCs localization within the architecture of the neurosphere. NSCs derived from neurospheres are not synchronized and are represented in all phases of the cell cycle. Cytofluorimetric studies demonstrate NSCs' heterogeneity in cell size by forward scatter (FSC) analysis, and in cytoplasmic granularity by side scatter (SSC) profiling. These findings may contribute to our understanding of the morphogenesis of the neurospheres, particularly as this process relates to the high environmental adaptability of the NSCs and the reported existence of different subpopulations of neural stem cells.