The replication of Epstein-Barr virus (EBV) and the expression of EBV early proteins were studied in the Burkitt's lymphoma cell line Akata stimulated with anti-human immunoglobulin G antibody (anti-IgG). Akata cells contained approximately 20 copies of EBV genome per cell as covalently closed, circular DNA. EBV DNA replication was observed at 6 hr and reached a maximal level at 24 hr after treatment with anti-IgG. Virion DNA was found in the culture medium at 12 hr. The kinetics of expression of BMRF1 gene product (early antigen diffuse component; EA-D) paralleled that of EBV deoxyribonuclease (DNase) and of DNA polymerase. Immunoblotting analysis showed that three polypeptides with molecular masses of 54, 52, and 49 kilodaltons (kDa) were recognized as EA-D components. The EBV DNase polypeptide was detected by immunoblotting at 53 kDa. The anti-EBV DNA polymerase antibody recognized 120- and 54-kDa polypeptides in the Akata cells. Immunoprecipitation followed by immunoblotting showed that EA-D and EBV DNase polypeptides were coimmunoprecipitated with anti-EBV DNase antibody and with anti-EA-D monoclonal antibody. These findings indicate that EA-D forms a complex with EBV DNase polypeptide. The molecules of EA-D, EBV DNase, and DNA polymerase appear to be closely associated together on the EBV replication.