Transmissible spongiform encephalopathies (TSE) arise as a consequence of the infection by prions of the central nervous system and are incurable. Neuronal loss and gliosis, associated with the accumulation of misfolded PrP protein (PrPSc) are hallmarks of prion diseases, however the mechanisms underlying these disorders remain unclear. We recently described a cellular model consisting of cerebellar granule cells derived from transgenic mice expressing ovine PrP. Upon exposure to low infectious doses of natural sheep scrapie agent, such cultures were found to accumulate de novo PrPSc and infectivity, as assessed by protease digestion and mouse bioassay. Both neurons and astrocytes were found to sustain prion propagation, and a late neuronal death was observed in infected cultures (1). In an attempt to generalize these observations, we found that it is also feasible to propagate mouse, hamster and human prions in cultures derived from transgenic mouse lines expressing the cognate PrPc. Primary cell cultures are widely used to search for molecules with protecting activity in neurodegenerative or neuro-infectious diseases as well as to assess their neurotoxicity. Thus we sought to evaluate the activity of Congo red, chlorpromazine and a polyene antibiotics, three compounds known to cure prion-infected cell lines, in these newly developed cell systems. We observed that the anti-prion activity in such cultures is species- or strain-dependent, and is in good agreement with results reported in vivo in rodent models. Our findings suggest that prion-infected primary neuronal cultures may be relevant tools for anti-prion drug appraisal, including human TSE agents.