A method which allows direct cloning of intracellular substrates for receptor tyrosine kinases (RTKs) was developed. By applying this technique to the study of the epidermal growth factor receptor (EGFR) signaling pathway, we have isolated a cDNA, designated eps8, which predicts a approximately 92 kDa protein containing an SH3 domain. Eps8 also contains a putative nuclear targeting sequence. Antibodies specific to the eps8 gene product recognize a protein of M(r) 97 kDa and a minor 68 kDa component, which are closely related, as demonstrated by V8 proteolytic mapping. The product of the eps8 gene is tyrosine-phosphorylated in vivo following EGF stimulation of intact cells and associates with the EGFR, despite the lack of a functional SH2 domain. Several other RTKs are also able to phosphorylate p97eps8. Thus, the eps8 gene product represents a novel substrate for RTKs. Adoptive expression of the eps8 cDNA in fibroblastic or hematopoietic target cells expressing the EGFR resulted in increased mitogenic response to EGF, implicating the eps8 gene product in the control of mitogenic signals.