The Src-like protein-tyrosine kinase Fyn is associated with T-cell antigen receptor. Transient expression of actively mutated Fyn, having Phe-528 instead of Tyr-528 or Thr-338 instead of Ile-338, in Jurkat T-cells stimulated the serum response element (SRE), 12-O-tetradecanoyl-phorbol-13-acetate response element, cyclic AMP response element, and c-fos promoter. The stimulation of SRE was particularly prominent not only with active Fyn but also with normal (wild-type) Fyn. SRE was also stimulated by both normal and active Lck. Furthermore, normal and active Fyn stimulated transcription from the IL-2 gene promoter when transfected cells were stimulated by concanavalin A plus 12-O-tetradecanoylphorbol-13-acetate. Under the same conditions, Lck did not stimulate IL-2 promoter unless it was activated by mutation. Interestingly, a mutant Fyn, which has deletions within the SH2 region and so is able to transform chicken embryo fibroblasts, did not stimulate either the c-fos or IL-2 promoter, suggesting the importance of this region in T-cell signaling. Csk, which phosphorylates tyrosine residues in the negative regulatory sites of Src family kinases, down-regulated Fyn- and Lck-mediated stimulation of the serum response element and Fyn-mediated enhancement of IL-2 promoter activity. These data suggest that Fyn and Lck, whose activities are regulated by Csk, are involved in different phases of T-cell activation.