The effects of chemical induction of latent Epstein-Barr virus (EBV) with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and n-butyrate on cell viability and induction of latent EBV in Raji and X50-7 B lymphocytes, indicated by expression of the diffuse component of the EBV early antigen (EA-D), were measured by visual immunofluorescence microscopy (of both viable and nonviable cells) and fluorescence-activated cell sorter (FACS) flow cytometry (of viable cells only). Cell viability at 4 days decreased moderately for treated Raji cells (9 to 37%, compared to 55 to 69% for untreated cells) and markedly for X50-7 cells (1-32% compared to 35-44% in untreated cells). The highest EA-D levels in viable cells occurred in Raji cells treated with both TPA and n-butyrate and untreated X50-7 cells. TPA and n-butyrate acted synergistically to induce latent EBV, resulting in increased levels of EA-D production in Raji cells and cell death in X50-7 cells. Methodological differences including the ability to detect antigen in only viable cells by FACS flow cytometry accounted for the higher levels of EA-D observed by FACS analysis compared to the levels observed by immunofluorescence microscopy. FACS analysis may be more objective and reproducible than immunofluorescence microscopy for the detection of EBV induction and also permits viral protein expression to be distinguished in the subpopulation of viable cells.