We have cloned two novel, alternatively spliced messages of human cyclin D-binding Myb-like protein (hDMP1). The known, full-length protein has been named hDMP1alpha and the new isoforms, hDMP1beta and hDMP1gamma. The hDMP1alpha, -beta, and -gamma splice variants have unique expression patterns in normal hematopoietic cells; hDMP1beta mRNA transcripts are strongly expressed in quiescent CD34+ cells and freshly isolated peripheral blood leukocytes, as compared with hDMP1alpha. In contrast, activated T-cells and developing myeloid cells, macrophages, and granulocytes express low levels of hDMP1beta transcripts, and hDMP1gamma is ubiquitously and weakly expressed. Mouse Dmp1 has been shown to activate CD13/aminopeptidase N (APN) and p19ARF gene expression via binding to canonical DNA recognition sites in the respective promoters. Assessment of CD13/APN promoter responsiveness demonstrated that hDMP1alpha but not hDMP1beta and -gamma, is a transcriptional activator. Furthermore, hDMP1beta was found to inhibit the CD13/APN promoter transactivation ability of hDMP1alpha. Stable, ectopic expression of hDMP1beta and, to a lesser extent hDMP1gamma, reduced endogenous cell surface levels of CD13/APN in U937 cells. Moreover, stable, ectopic expression of hDMP1beta altered phorbol 12-myristate 13-acetate-induced terminal differentiation of U937 cells to macrophages and resulted in maintenance of proliferation. These results demonstrate that hDMP1beta antagonizes hDMP1alpha activity and suggest that cellular functions of hDMP1 may be regulated by cellular hDMP1 isoform levels.