We previously reported that BARF1 gene has either an immortalizing effect, when expressed in primary primate epithelial cells, or a malignant transforming activity, when expressed in established and nontumoral rodent fibroblast or human B-cell lines. As predicted from sequence analysis, we found that BARF1 coded protein can be secreted from different cell lines, among them BARF1-transfected Balb/c3T3 rodent fibroblasts. Thus, as an initial step to clarify BARF1 oncogenic functions, we investigated whether the secreted form of BARF1 protein can activate the cell cycle as a growth factor. Since efficient BARF1 expression could be obtained from 293-tTA cells infected with a tetracycline-regulatable recombinant adenovirus, secreted BARF1 product could be purified from the culture medium of such cells by ammonium sulfate precipitation, ion exchange chromotography and sucrose gradient sedimentation. We describe in this paper that addition of a purified product of secreted BARF1 protein to serum-free culture medium of Balb/c3T3 rodent fibroblasts, human Louckes B-cell line and primary monkey kidney epithelial cells resulted in a cell cycle activation that was inhibited by affinity-purified anti-BARF1 antibody. Our demonstration of a specific stimulation of cell cycle in vitro by BARF1 secreted product suggests that this EBV-encoded BARF1 protein could act as a growth factor in vivo.