The risk of contamination of tissue culture cells with transmissible spongiform encephalopathy (TSE) agents as a result of the use of animal products as medium components has been considered to be low, in part, because only a few brain-derived cell lines have been reported to be susceptible to TSE infection. In the present study, we demonstrate that the common laboratory fibroblast cell lines NIH/3T3 and L929, which express low levels of cellular mouse prion protein, are susceptible to infection with mouse-adapted scrapie. Our results show that the susceptibility of a cell line to TSE infection cannot be predicted on the basis of its tissue origin or its level of expression of the cellular prion protein, and they suggest that any cell line expressing normal host prion protein could have the potential to support propagation of TSE agents. Thus, testing of cells for TSE susceptibility might be necessary for all cell lines that are routinely used in vaccine production and in other medical applications.