Evi9, a common site of retroviral integration in BXH2 murine myeloid leukemias, encodes a C2H2 zinc finger protein and is overexpressed in these leukemic cells. To investigate a possible role of EVI9 in the human hematopoietic system, we isolated the cDNA clone of the human homologue. Human EVI9, located on the chromosome 2p13 region, contains an open reading frame of 797 amino acids that is 98.7% identical to the mouse protein. RT-PCR analysis of purified human hematopoietic cells showed that EVI9 is expressed in CD34-positive myeloid precursors, B cells, monocytes, and megakaryocytes, but only weakly in T lymphocytes, suggesting that EVI9 may play an important role in hematopoiesis. Furthermore, EVI9 was down-regulated during myeloid differentiation of HL60 cells induced by all-trans-retinoic acid, whereas the expression remained during monocytic differentiation induced by phorbol 12-myristate 13-acetate. These results indicate a distinct role for EVI9 in human hematopoietic cells and suggest that EVI9 may cause leukemia through inhibition of myelold differentiation.