Only a few cell lines have been infected with prions, offering limited genetic diversity and sensitivity to several strains. Here we report that cultured neurospheres expressing cellular prion protein (PrP(C)) can be infected with prions. Neurosphere lines isolated from the brains of mice at embryonic day 13-15 grow as aggregates and contain CNS stem cells. We produced neurosphere cultures from FVB/NCr (FVB) mice, from transgenic (Tg) FVB mice that overexpress mouse PrP-A (Tg4053), and from congenic FVB mice with a targeted null mutation in the PrP gene (Prnp(0/0)) and incubated them with the Rocky Mountain Laboratory prion strain. While monitoring the levels of disease-causing PrP (PrP(Sc)) at each passage, we observed a dramatic rise in PrP(Sc) levels with time in the Tg4053 neurosphere cells, whereas the level of PrP(Sc) decayed to undetectable levels in cell cultures lacking PrP. PrP(Sc) levels in cultures from FVB mice initially declined but then increased with passage. Prions produced in culture were transmissible to mice and produced disease pathology. Intracellular aggregates of PrP(Sc) were present in cells from infected cultures. The susceptibility of neurosphere cultures to prions mirrored that of the mice from which they were derived. Neurosphere lines from Tg4053 mice provide a sensitive in vitro bioassay for mouse prions; neurosphere lines from other Tg mice overexpressing PrP might be used to assay prions from other species, including humans.