Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high-affinity binding sites for cellular proteins containing Src homology 2 (SH2) domains. These proteins transduce various mitogenic and morphogenic signals. As reported previously, Y696KNI in the kinase insert domain of v-Fms binds to the growth factor receptor bound protein 2 (Grb2), a stimulator of the Ras/Raf1 pathway. Here, we mapped Y921TNL within the C-terminal domain of Fms as a novel autophosphorylation site. We demonstrate that this site constitutes a second Grb2 binding site: a recombinant fusion protein (residues 904-944) containing phosphorylated Y921 bound Grb2 from FDCP-1Mac11 cell extracts significantly more efficiently than a corresponding protein (residues 617-759) containing Y696. A yeast two-hybrid system which allowed the formation of a functional Fms tyrosine kinase was employed to quantify binding of Grb2. Fms-protein containing either one of the two phosphorylation sites bound Grb2 equally well, binding was increased for proteins carrying both sites. In contrast, the simultaneous substitution of Y696 and Y921 by phenylalanines abolished Grb2 binding. Mouse NIH3T3 cells expressing the Y921F mutant Fms-protein showed a substantially higher content of fibronectin network than wild-type transformed cells and had largely lost their serum independent growth phenotype.