To study the role of p59fyn in T cell activation, we used antisense RNA to inhibit p59fyn expression in a T cell clone. Transfectants with reduced levels of p59fyn were functionally impaired in their responses to antigen, Con A + recombinant IL-1 and cross-linking with anti-TCR mAb. Induction of tyrosine phosphorylation on most intracellular substrates was greatly reduced. We also noted that the Ick kinase activity was greatly reduced even though the amount of Ick protein was equivalent to that present in parental D10 cells. Our results suggest that the protein tyrosine kinase p59fyn is critical in TCR-mediated signaling and also suggests that p59fyn may regulate p56fyn tyrosine kinase activity.