Two monoclonal antibodies (mAb) were derived and designated 4F10 and 31 IH. 4F10 was against the Epstein-Barr virus (EBV) Zta protein and 311 H specifically recognized EBV DNase enzyme. Using mAb 4F10 as a probe, the Zta protein could be detected as a 36-kD molecule in L5 cells and as a 38-kD molecule in B95-8 cells, reflecting the fact reported by other laboratories, using rabbit polyclonal antisera, that the Zta protein was variously modified in different host cells. 311H mAb was generated using antigens purified from onestep His-Bind column chromatography. The antigenic epitope recognized by this mAb was mapped within the residues 1-152 of EBV DNase by reacting the mAb with three distinct truncated mutants. Also, using 311H as a reagent to trace the kinetic expression of EBV DNase proteins in EBV-infected Akata cells, the Western blotting results indicated that DNase antigen could be detected at 12 h postactivation. The feasibility of applying these two mAb in the investigation of EBV biology is discussed.