Subgroup C strains of Herpesvirus saimiri, a leukemogenic virus of non-human primates, transform human T cells to permanent growth in culture. These cell retain their antigen specificity, and they are becoming widely used as a model for activated human T cells. Though a variety of human cell types can be infected by H. saimiri, transformation appears to be specific for CD4+ and CD8+ T cells. Our investigation of early signaling events in H. saimiri-transformed T cells revealed a novel 40-kDa phosphoprotein complexed with the T cell-specific tyrosine protein kinase p56lck. This protein, termed Tip (tyrosine kinase interacting protein), is identified as a viral protein encoded by the open reading frame 1 (ORF1). In the transformed cells Tip is expressed together with the gene product of ORF2, the viral oncoprotein StpC, which acts on epithelial cells. The H. saimiri genome has 75 ORFs, but only ORF1 and ORF2 are transcribed in transformed human cells. Tip is phosphorylated on tyrosine in cell-free systems containing Lck, indicating that the viral protein is a substrate for this T cell-specific kinase. Alteration of T cell signaling pathways by Tip may be the second event complementing the action of StpC in a new mechanism of T cell transformation.