Grb2/Ash is composed of one SH2 and two SH3 domains and functions as an adapter linking tyrosine kinase receptors and Ras in fibroblasts. We have investigated the nature of proteins interacting with Grb2/Ash in hematopoietic cells. The product of the vav proto-oncogene (Vav) is expressed exclusively in hematopoietic cells and has guanine nucleotide exchange activity. Here we report that granulocyte/macrophage-colony- stimulating factor (GM-CSF), interleukin-3, and erythropoietin (Epo) induce rapid and transient tyrosine phosphorylation of Vav and that Vav is constitutively associated with the SH3 domain of Grb2/Ash in a human leukemia cell line UT-7. These data implicate Vav in a signaling pathway leading to activation of Ras or Ras-related proteins in hematopoietic cells. Furthermore, we have shown that the proto-oncogene c-cbl product (c-Cbl) is also tyrosine-phosphorylated by stimulation with GM-CSF or Epo and is constitutively associated with the SH3 domain of Grb2/Ash in UT-7. However, we could not find the homologous regions with guanine nucleotide exchange factors or GTPase-activating proteins in the c-cbl gene. Therefore, Grb2/Ash might also transduce a signal that is different from the signal leading to the small-G protein regulation.