Immortalized bovine brain cell lines provide ideal in vitro cellular infection models for bovine spongiform encephalopathies (BSEs) caused by prions without enduring species barrier. We have established an immortalized brain cell line (FBBC-1 cells) from primary cultures of cryopreserved fetal bovine brain tissues after transfection with SV40 large T antigen. FBBC-1 cells are stable after passaging to >100 population doublings after single cell cloning, with a generation time of 24 h. After the treatment with dibutyryl-cyclic AMP, the cells ceased proliferation and extended neurite-like processes that were immunostained with the antibody against tubulin βIII, a marker of immature neurons. Upregulation of tubulin βIII expression was confirmed by immunoblotting. These bovine cells expressed cellular prion protein and its processed smaller C1 fragment, and may provide an in vitro means of propagating cattle BSE prion.