Thymosin beta 4 (T beta 4) was originally isolated as a thymic hormone. Its functional properties remain obscure; however, the N-terminal peptidic sequence could have a regulatory function on hematopoietic stem cell proliferation. To investigate the mechanism of T beta 4 expression, we studied T beta 4 gene expression in various leukemic cells and in established cell lines. Among leukemic cell samples obtained from leukemia patients, the T beta 4 gene was highly expressed in a lymphoid lineage, especially in adult T-cell leukemia (ATL) cells, rather than in a granulocyte lineage. The T beta 4 gene was more transcriptionally active in chronic B-cell leukemia than in acute B-cell leukemia, while it was inactive in plasma cell leukemia. We also found that cells from one of the ATL patients transcribed a heterogeneous message. T beta 4 messenger RNA increased in MOLT-3 during differentiation by 12-O-tetradecanoylphorbol-13-acetate (TPA), in HL60 cells induced by TPA or dimethylsulfoxide and K562 cells stimulated by cytosine arabinoside or hemin. The genomic sequence of T beta 4 is considered to be highly conserved. Only 1 of 20 genomes from normal or hematopoietic malignant cells showed restriction fragment length polymorphism. These findings, along with previous data, suggest that T beta 4 may be a new marker of differentiation of hematopoietic cells.