Cell cultures represent relevant and useful experimental models of transmissible spongiform encephalopathies (TSEs). They are able to promote, upon subpassaging, stable and persistent replication of PrP(Sc) as well as infectivity. To date, only a few cell culture models permissive to prion replication are available. Among them, mouse neuroblastoma cell lines (N2a) are most commonly used. While they correspond to homologous models supporting propagation of mouse-adapted scrapie strains, recent studies have reported heterologous models sensitive to natural occurring disease. Infected cell culture models have provided some valuable insights into the biogenesis of PrP(Sc) in terms of conversion, subcellular localisations, physiopathological consequences and species-barrier determinants. They have also contributed to the screening and the study of possible therapeutic compounds and to the development of new strategies for the investigation of TSE-specific diagnostic markers.