The Tec family of protein tyrosine kinases plays an important role in T cell signaling. Tec, the prototypical member of this kinase family, can interact with CD28, which is a costimulatory molecule. However, the regulation of Tec upon CD28 stimulation remains poorly understood. Here we show that CD28-B7-mediated interactions are likely involved in the relocalization of Tec at the contact zone between T cells and APC. Upon CD28 ligation with specific antibodies or natural ligands, Tec translocates to the plasma membrane where it colocalizes with the CD28 molecule. The Src-homology 3(SH3) domain of Tec and the two proline-rich motifs of CD28 are involved in this process. Furthermore, we show that CD28 signaling requires the SH3 domain of Tec as well as proline residues present in the intracytoplasmic tail of CD28. These results should provide new insights into the complex regulation of Tec kinases in T cells.