Previous work from our laboratory has demonstrated the rapid phosphorylation on tyrosine of a 66-kDa soluble protein (TPP 66), when human T lymphocytes were incubated with the mitogenic lectins phytohemagglutinin or concanavalin A. To further explore the role of TPP 66 in lymphocyte activation we have utilized a variety of agents which either activate directly or augment the activation of the T cell and assayed their effect on TPP 66 phosphorylation. Wheat germ agglutinin, the divalent cation ionophore A23187, and phorbol myristate acetate have been demonstrated to activate human T cells and each of these agents also induced the rapid phosphorylation of TPP 66. In addition, epidermal growth factor and platelet-derived growth factor also induced the phosphorylation of TPP 66. Phosphorylation was seen at concentrations that are within the active dose range for all agents tested. Phosphorylation was on tyrosine, since base hydrolysis of TPP 66 followed by single-dimension high-voltage electrophoresis revealed radioactivity only in the area corresponding to authentic phosphotyrosine. These data demonstrate that a variety of agents which either directly activate human T lymphocytes or augment activation are associated with the tyrosine phosphorylation of TPP 66 and provide further evidence for a critical role for this protein in the control of lymphocyte activation.