The BNLF-1 protein is the only non-nuclear Epstein-Barr virus (EBV) encoded protein that has been detected in B-lymphocytes immortalized by EBV. We demonstrate that the BNLF-1 gene induces anchorage-independent growth and tumorigenic transformation of the murine cell-line, Balb/3T3. This demonstration extends the earlier observation that the BNLF-1 gene can transform Rat-1 cells. In addition we find that the BNLF-1 protein is located in the particulate fraction of cells, is phosphorylated, and is turned over with a half-life of 2.0 to 3.5 h in the BNLF-1 transformed Balb/3T3 cells, just as it is in EBV-genome-positive B-cell-lines.