The protein-tyrosine kinase Csk is one of the main down-regulators of the Src family of kinases. Csk may be involved in the down-regulation of T cell receptor (TCR) signaling by C-terminal tyrosine phosphorylation of Lck and Fyn; however, it is not known how Csk activity is regulated or how it targets these Src family members. We used Jurkat T cells and normal human T cells to examine proteins that bind to the SH2 domain of Csk. In both Jurkat and normal T cells, the Src homology 2 (SH2) domain of Csk bound constitutively to a tyrosine-phosphorylated protein of 60 kDa (p60). The 60-kDa protein was detected in Csk immunoprecipitates from both unstimulated and CD3-stimulated cells. In addition to p60, a protein of 190 kDa coprecipitated with Csk, and both proteins were phosphorylated on tyrosine residues by the immunocomplex. Small amounts of GTPase-activating protein (GAP) were detected in anti-Csk immunoprecipitates, suggesting that p60 may be a GAP-associated protein. Our data demonstrate that the SH2 domain of Csk specifically associates with at least two tyrosine-phosphorylated proteins in normal human T cells, that this association is independent of TCR/CD3 activation, and that Csk may be a part of a multiprotein complex containing GAP.