The src family protein tyrosine kinases participate in signaling through cell surface receptors that lack intrinsic tyrosine kinase domains. One of the src family kinases, p59fyn (Fyn), plays an important role in the TCR-mediated signaling. Here we report that Fyn becomes associated with the zeta-associated tyrosine kinase, ZAP-70, in a T cell hybridoma upon stimulation. The association was transient; it occurred as early as 10 s after stimulation and disappeared after 10 min. The two proteins were also associated with each other when coexpressed in COS cells. Coexpression of the zeta-chain was not required for their interaction. Mutational analysis of Fyn and ZAP-70 revealed that their kinase activities were relevant to the association. Deletion of both the SH2 and SH3 domains of Fyn resulted in the decrease of the association with ZAP-70. Consistently, Fyn-SH2 and Fyn-SH3 fused to glutathione S-transferase were able to bind to ZAP-70. These data suggest that multiple sites of Fyn and ZAP-70 are involved in the association. Furthermore, coexpression of the wild-type of both kinases in COS cells enhanced tyrosine phosphorylation of the helix-turn-helix-containing protein, HS1. HS1 was also tyrosine phosphorylated upon TCR stimulation. Thus, we propose that Fyn phosphorylates and activates ZAP-70 and that both kinases cooperate in TCR signaling.