Using a combination of immunofluorescence and autoradiography, we studied the appearance of EBNA and DNA synthesis in cord-blood lymphocytes after infection with EBV derived from the B95-8 cell line. EBNA appeared between 12 and 25 h after addition of the virus. DNA synthesis was detected in EBNA-positive cells approximately 20 h after the appearance of EBNA. This shows that EBNA induction precedes the first cellular S-phase and suggests that the cells have not yet entered the cell-division cycle when EBNA appears. Little, if any, of the total DNA synthesis induced at this stage can be attributed to EBV-mediated immunologic stimulation.