We report a new rodent cell-transforming gene, presumably involved in viral replication, encoded by Epstein-Barr virus. We previously showed that the corresponding open reading frame BARF1 is transcribed before the onset of viral DNA synthesis, and translated into a 33 kd early polypeptide (p33). Here we show that recombinant plasmids containing the BARF1 induce morphological change, anchorage-independent growth and tumorigenic transformation of established mouse fibroblast lines. The BARF1-transformed cells and the tumour tissues isolated from new-born rats after injection of such transformed cell both express p33. Transforming activity was obtained from either the genomic fragment or the cDNA sequence.