Previous evidence indicated that frog virus 3 (FV3), an icosahedral DNA virus, replicates exclusively in the cytoplasm. However, data presented here demonstrate that FV3 does not replicate in UV-irradiated or enuleated chicken embryo or BSC-1 cells and that virus-specific DNA synthesis is not initiated in such cells. Primary transcription was not detected in infected enucleated cells. These results demonstrate that a functional nucleus is essential for FV3 replication.