Monoclonal antibodies (mAbs) were prepared against varicella-zoster virus (VZV)-infected cell proteins, and 10 mAbs which reacted with nuclear antigens were selected. These mAbs recognized a major 175-180 kDa and three minor VZV-specific phosphoprotein species. Immunofluorescence staining of VZV-infected cells showed that the 175-180 kDa protein was synthesized within 6 h after infection. The synthesis of this protein was inhibited by cycloheximide (CH); however, reversal of CH treatment and addition of actinomycin D (ActD) resulted in the synthesis of the 175-180 kDa protein. To determine whether the 175-180 kDa protein seen in the infected cells is encoded by VZV immediate early (IE) gene 62, the predicted open reading frames of VZV genes 61 and 62 were cloned into pGEM transcription vectors. RNA was transcribed from each gene, translated in vitro and immunoprecipitated with a mAb which recognizes a major 175-180 kDa and three minor proteins. The reactivity of the in vitro translation products encoded by gene 62 with this mAb suggested that the 175-180 kDa protein is encoded by VZV IE gene 62.