The intracellular expression of two early gene products of Epstein-Barr virus (EBV) was evaluated by fluorescence flow cytometry. Two Burkitt's lymphoma cell lines were superinfected by EBV from the P3HR-1 cell line. Twelve to 96 hr after superinfection the cells were fixed with paraformaldehyde and made permeable by saponin treatment. Monoclonal antibodies to the diffuse (D) and restricted (R) components of the early antigen (EA) complex were applied to the cells followed by fluorochrome-conjugated goat antibodies specific for the heavy chain isotypes of the monoclonal antibodies. Fluorescence flow cytometry revealed a clear separation in fluorescence intensity between cells containing EA-R or EA-D and negative cells. A major proportion of EA-positive cells displayed both antigens. In addition, a significant fraction expressed either EA-R or EA-D but not both. Expression of EA occurred more rapidly and peaked earlier in Daudi cells than in Raji cells. This apparent difference in EA expression between the two cell lines, however, was much less pronounced when one considered the proportion of cells expressing EA-R only, EA-D only, or both EA-R and EA-D, as a percentage of the total EA expression. Parallel fluorescence microscopy experiments revealed that the variation of the ratio of EA-R to EA-D expression with time correlated well between the two methods.