A new medium, PPRF-m2, has been developed for the in vitro expansion of murine neural stem cells. Neural stem cells (NSC) are primitive cells that have the capability to give rise to all of the cells that comprise the central nervous system (CNS). PPRF-m2 was developed in part by examining key aspects of a commercial NSC medium used in our laboratory, and includes the basal media combination RPMI/DMEM/F12 in a 1:I:1 volumetric ratio supplemented with a hormone mixture, sodium bicarbonate (21.4 g/L), Hepes (4.3 g/L), glucose (1.75 g/L), glutamine (0.14 g/L), epidermal growth factor (20 mu g/L) and basic fibroblast growth factor(10 mu g/L). Cells inoculated into PPRF-m2 effectively doubled 4 times in 5 days to achieve a maximum viable cell density of 1.2 x 10(6) cells/mL in stationary culture. This was approximately 650% higher than the maximum cell density achieved in the commercial NSC medium. In addition, the PPRF-m2 cultures were observed to contain less cellular debris, and the cells were less granular and exhibited a lower degree of attachment. Immunocytochemistry revealed that cells grown in PPRF-m2 retained the ability to generate neurons, astrocytes, and oligodendrocytes.