p50(csk) is a cytosolic tyrosine protein kinase expressed in all cell types. Accumulating data show that it inhibits multiple cellular processes, as a consequence of its ability to repress the enzymatic activity of Src family tyrosine protein kinases. We previously demonstrated that, via its Src homology 3 (SH3) domain, Csk is tightly bound to PEP, a protein-tyrosine phosphatase (PTP) exclusively expressed in hemopoietic cells. In this report, we have tested the possibility that Csk also interacts with PTP-PEST, a ubiquitous PTP sharing structural homology with PEP. Our studies revealed that Csk was associated with PTP-PEST in a variety of cell types, including non-hemopoietic cells. This interaction involved the SH3 region of p50(csk) and a proline-rich region (PPPLPERTPESFVLADM) outside the catalytic region of PTP-PEST. Even though both PTP-PEST and PEP were associated with Csk, significant differences were noted between these two PTPs. PTP-PEST, but not PEP, was also complexed with Shc, an adaptor molecule implicated in the Ras pathway. Moreover, PTP-PEST and PEP were found to accumulate primarily in distinct intracellular compartments in cell fractionation studies. In combination, these findings indicated that, like PEP, PTP-PEST is probably involved in Csk-mediated functions in mammalian cells. Moreover, they suggested that the roles of Csk-PTP-PEST and Csk-PEP are likely to be different.