Herpesvirus saimiri (HVS) of subgroup C efficiently induces leukemia in New World primates and transforms human lymphocytes. The viral tyrosine kinase interacting protein (Tip) binds to the tyrosine protein kinase Lck and is essential for transformation. Understanding how Tip modulates Lck activity is important for elucidating the mechanism of herpesvirus saimiri leukemogenesis. However, there are reports suggesting that whereas the Tip protein of HVS strain 484 stimulates the activity of Lck, the Tip protein of HVS strain 488 inhibits Lck. To determine whether these two divergent Tip proteins have opposite effects on Lck activity, we compared them in parallel. We found that both Tip proteins stimulated Lck kinase activity in vivo and in vitro and that both stimulated NF-AT- and STAT3-dependent transcription in T cells. Our data support the model that HVS infection increases the activity of Lck through the action of Tip.