The t(9;22) chromosomal translocation is found in almost all patients with chronic myelogenous leukemia. The resultant Bcr-Abl fusion gene expresses a chimeric fusion protein p210bcr-abl with increased tyrosine kinase activity. Hematopoietic progenitors isolated from chronic myelogenous leukemia patients in the chronic phase contain constitutively tyrosine-phosphorylated p62dok protein. p62dok associates with the Ras GTPase-activating protein (RasGAP), but only when p62dok is tyrosine phosphorylated. Here we have investigated the interaction between p62dok and RasGAP and the consequences of p62dok tyrosine phosphorylation on the activity of RasGAP. We have found that p62dok is directly tyrosine phosphorylated by p210bcr-abl, and the sites of phosphorylation are located in the C-terminal half of the p62dok molecule. We have identified five tyrosine residues that are involved in in vitro RasGAP binding and have found that tyrosine-phosphorylated p62dok inhibits RasGAP activity. Our results suggest that p210bcr-abl might lead to the activation of the Ras signaling pathway by inhibiting a key down-regulator of Ras signaling.