We isolated a cDNA clone encoding a novel Src homology (SH)2 domain-containing protein of 47 kDa from a human cDNA library. As its transcript was predominantly expressed in hematopoietic cells, this gene was termed HSH2 for hematopoietic SH2 protein. This protein contains several putative protein-binding motifs, SH3-binding proline-rich regions, and phosphotyrosine sites, but lacks enzymatic motifs. In a yeast two-hybrid screen, we identified a cytokine-regulated tyrosine kinase c-FES and an activated Cdc42-associated tyrosine kinase ACK1 as HSH2 interactors. HSH2 bound c-FES via its C-terminal region as well as its N-terminal region including the SH2 domain, whereas it bound ACK1 via its N-terminal proline-rich region. Furthermore, these two kinases bound and tyrosine-phosphorylated HSH2 in mammalian cells. Hence, we postulate that HSH2 functions as an adapter protein involved in tyrosine kinase signaling, and possibly regulates cytokine signaling and cytoskeletal reorganization, in hematopoietic cells.