Neural stem cells (NSCs) are undifferentiated, primitive cells with important potential applications including the replacement of neural tissue lost due to neuro-degenerative diseases, including Parkinson's disease, as well as brain and spinal cord injuries, including stroke. We have developed methods to rapidly expand populations of mammalian stem and progenitor cells in neurosphere cultures. In the present study, flow cytometry was used in order to understand cell cycle activation and proliferation of neural stem and progenitor cells in suspension bioreactors. First, a protocol was developed to analyze the cell cycle kinetics of NSCs. As expected, neurosphere cells were found to cycle slowly, with a very small proportion of the cell population undergoing mitosis at any time. Large fractions (65 - 70%) of the cells were detected in G1, even in rapidly proliferating cultures, and significant fractions (20%) of the cells were in GO. Second, it was observed that different culturing methods influence both the proportion of neurosphere cells in each phase of the cell cycle and the fraction of actively proliferating cells. The results show that suspension culture does not significantly alter the cell cycle progression of neurosphere cells, while long-term culture (>60 days) results in significant changes in cell cycle kinetics. This suggests that when developing a process to produce neural stem cells for clinical applications, it is imperative to track the cell cycle kinetics, and that a short-term suspension bioreactor process can be used to successfully expand neurosphere cells. (C) 2004 Wiley Periodicals, Inc.