In this study we compare the effect of CD3 and CD2 ligation on tyrosine kinase activation in human peripheral blood T cells. Using antiphosphotyrosine antibody to detect tyrosine phosphorylation of cellular substrates, we demonstrate that mAb stimulation of either CD3 or CD2 results in tyrosine phosphorylation of the TCR-zeta chain and 135- and 100-kDa proteins. However, differences are observed between CD3 and CD2 ligation; only the former results in rapid tyrosine phosphorylation of 72-, 65-, and 40-kDa substrates. Co-aggregation of CD2 and CD45, a tyrosine phosphatase, results in inhibition of intracellular calcium elevation and T cell proliferation. We demonstrate in this study that this manipulation also inhibits polyphosphoinositide hydrolysis and tyrosine phosphorylation of the 100-kDa substrate. The failure of tyrosine phosphorylation of the 100-kDa substrate is specific in that phosphorylation of the 135-kDa protein is not inhibited. Similar results are observed when CD2 and CD45 are independently cross-linked rather than co-aggregated. The observation that CD45 cross-linking alters tyrosine phosphorylation of T cell substrates and effects polyphosphoinositide hydrolysis is further evidence that tyrosine phosphorylation regulates early events in T cell activation including, perhaps, phospholipase C activity.