Identification of physiological substrates of protein tyrosine phosphatases is a key step in understanding the function of these enzymes. We have generated fibroblast cell lines having a gene-targeted PTP-PEST in order to identify potential substrates with the premise that specific substrates of this enzyme would exist in a hyperphosphorylated state. Analysis of the profile of the phosphotyrosine proteins in the PTP-PEST -/- cells revealed the presence of hyperphosphorylated proteins of 180, 130, and 97 kDa when compared to control cells. The p130 was identified as p130(Cas), and direct immunoprecipitates of p130(Cas) demonstrate that this protein is constitutively hyperphosphorylated in cells lacking PTP-PEST. In addition, p130(Cas) was also isolated by the substrate-trapping mutant of PTP-PEST in the PTP-PEST -/- cell lysates. Interestingly, we have demonstrated for the first time that PTP-PEST, through its first proline-rich sequence 332PPKPPR337, interacts with other members of the p130(Cas) family (Hef1 and Sin) via their SH3 domain in vitro. This result suggests that Hef1 and Sin could also be potential substrates of PTP-PEST. In conclusion, we have combined genetic and biochemical strategies to allow the identification of PTP-PEST substrates. This experimental approach could potentially be used to identify substrates of other PTPases. Furthermore, the Cas-like molecules Hef1 and Sin associate via their SH3 domains with a proline-rich motif found on PTP-PEST, suggesting the possibility that PTP-PEST could be a general modulator of the Cas family of proteins.