From an early stage of prion research, tissue cultures that could support and propagate the scrapie agent were sought after. The earliest attempts were explants from brains of infected mice, and their growth and morphological characteristics were compared with those from uninfected mice (1). Using the explant technique, several investigators reported increased cell growth in cultures established from scrapie-sick brain compared with cultures from normal mice (1, 2). These are odd findings in the light of the massive neuronal cell death known to occur in scrapie-infected brains; however, the cell types responsible for the increased cell growth in the scrapie-explants most probably were not neuronal. The first successful cell culture established in this way, in which the scrapie agent was serially and continuously passaged beyond the initial explant, was in the scrapie mouse brain culture (3), which is still used today (4, 5). This chapter describes the generation and use of chronically prion-infected cell lines as cell culture models of prion diseases. These cell lines have been crucial for the current understanding of the cell biology of both the normal (PrP^C) and the pathogenic isoform (PrP^Sc) of the prion protein. They also have been useful in the development of antiprion drugs, prospectively used for therapy of prion diseases, and they offer an alternative approach for transmission/infectivity assays normally performed by mouse bioassay. Cell culture models also have been used to study prion-induced cytopathological changes, which could explain the typical spongiform neurodegeneration in prion diseases.