Latent Epstein-Barr virus (EBV) infection activates B-lymphocyte proliferation through mechanisms which are partially known. One approach to further delineate these mechanisms is to identify cellular genes whose expression is augmented in cells latently infected with EBV. Since EBV-negative Burkitt's lymphoma cells can be grown in continuous culture and EBV can establish growth-altering latent infection in these cells, some effects of EBV on B-lymphocyte gene expression can be studied by using this in vitro system. Pursuing this latter approach, we have used cDNA cloning and subtractive hybrization to identify a gene whose expression is increased after EBV infection. This gene encodes the cytoskeletal protein vimentin. Latent infection of established EBV-negative Burkitt's lymphoma cell lines with the transforming EBV strain, B95-8, resulted in dramatic increases in vimentin mRNA and protein levels, while infection with the nontransforming P3HR1 strain failed to do so. Vimentin induction was reproduced by the expression of the single EBV gene which encodes the latent infection membrane protein (LMP). An amino-terminal LMP deletion mutant did not induce vimentin. These results are of particular interest in light of the transforming potential of LMP, as demonstrated in rodent fibroblasts, and the interaction between vimentin and LMP observed in immunofluorescent colocalization and cell fractionation studies.