Herpesvirus saimiri (HVS) is a T-cell-specific transforming and oncogenic virus. A protein encoded by HVS known as Tip-484 (for tyrosine kinase interacting protein from HVS strain 484) is required for this transformation, Tip-484 binds specifically to the nonreceptor protein tyrosine kinase p56lck. By transfecting Tip-484 into T cells, we now show that this interaction leads to a several hundred-fold increase in the kinase activity of p56lck. Tip-484 is part of a protein complex which is dependent on the presence of p56lck and is phosphorylated. We also show that two of the complexed proteins represent two phosphorylated forms of Tip-484. Furthermore, the p56lck kinase activity in HVS-infected human peripheral blood T lymphocytes was at least ninefold higher than that in noninfected control cells and significantly decreased in cells infected with a Tip-484 deletion mutant virus. Finally, we report that Tip-484 is required for oncogenesis in rabbits by the survival of rabbits inoculated with Tip-484 deletion mutant HVS. The data demonstrate dramatic stimulation of the signaling pathway of p56lck. This effect can contribute to the molecular mechanisms that lead to sustained autocrine secretion of growth factors, permanent T-cell growth, and ultimately lymphocytic tumor formation.