An image cytometry algorithm was developed to identify and quantify immunologically labeled cells in cultured human neurospheres. Image cytometry revealed new findings in terms of the localization of specific types of neural cells and the regional fluctuation of cell density in neurospheres. The distributions of total cell density exhibited similar profiles along the radial distance from the periphery of a neurosphere, irrespective of neurosphere sizes examined; approximately 1.0 x 10(6) cell/mm(3) in the vicinity of the periphery and 6.0 x 10(5) cell/mm(3) in an inner region of 50-180 microm. In addition, BrdU-positive (proliferating) cells were present at ratios of 53-64% across the entire region of the neurospheres, whereas BrdU-negative and beta III tubulin-positive cells (postmitotic neurons) were minorities in the neurospheres.