Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells does replicate in EC cells, although less efficiently than the DNA of PyEC mutants. This replication proceeds via a Tantigen-independent mechanism as confirmed by the behavior of a Py tsa mutant, which produces a thermosensitive large T antigen, of a deletion mutant which lacks part of the large T protein-coding sequence, and of a double mutant carrying both the tsa and the PyEC F9 mutations. We also show that the ability to replicate Py DNA in the absence of T antigen is lost when EC cells are allowed to differentiate. Finally, we discuss the involvement of both viral DNA sequences and cellular proteins in this phenomenon. MATERIALS AND METHODSCell cultures and virus strains. NIH 3T6 mouse cells and the EC cell lines PCC4-aza-1, F9, and LT1 (12,16,21) were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (GIBCO Diagnostics, Madison, Wis.). F9 cells were grown on gelatin-coated (0.1%) dishes. All virus strains were propagated by infecting secondary mouse embryo cells at a multiplicity of 0.01 PFU per cell. The wild-type Py used in these studies was the large plaque strain A2 (10). The nucleotide numbers refer to the Py DNA sequence as reported by Soeda et al. (19). The PyEC mutant PyF9-1 has been previously described (13