We constructed two reticuloendotheliosis virus (REV) envelope gene expression plasmids, one containing the REV-A envelope gene, the other the spleen necrosis virus (SNV) envelope gene. Cell lines were generated by transfecting each of the REV envelope plasmids into D17 cells, a canine cell line. The levels of REV envelope glycoprotein in the cell lines were assayed by immunoprecipitating the envelope glycoproteins from lysates of cells that were labeled with [35S]methionine. Virological challenge assays determined the degree of resistance of each of the cell lines to REV-A or SNV infection. The expression of either envelope gene protected the cells from infection by either REV-A or SNV virus. Several cell lines were significantly more resistant to REV infection than the parental D17 cells, and two lines were 25,000-fold more resistant, approaching the resistance of REV-infected D17 cells to reinfection. The resistant cell lines were not able to confer resistance to susceptible cells by cocultivation. The level of resistance was correlated with the uniformity of expression of the REV envelope glycoproteins by the individual cells in a cell line and not with the absolute level of expression by the population of cells.