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Two monoclonal antibodies (MAbs) were obtained from BALB/c mice immunized with cowpox virus (CPV)-infected cells cultured in the presence of cytosine-1-β-d-arabinofuranosyl-HCl (Ara C). In the immunofluorescence test, the specific antigen reacting with these MAbs was restricted almost entirely to the nucleus in CPV-infected cells in the presence of Ara C at any time. On the other hand, in CPV-infected cells in the absence of Ara C, the antigen was first detectable within the nucleus 2 h after infection and migrated to the cytoplasm as the infection proceeded. On immuno-blotting, only one component with an M r of 27K was detected 2 to 24 h after infection of cells with CPV in either the presence or absence of Ara C. The antigen was also detected in vaccinia virus-infected cells, but not in any mock-infected cells nor in any virions purified from infected cells. These results suggest that the antigen reacting with the MAbs is virus-specific and a non-structural early antigen.