The binding of the Myb-like DMP1 transcription factor to DNA consensus sequences [CCCG(G/T)ATGT] in artificial promoters is antagonized by D-type cyclins with no requirement for their catalytic partners, cyclin-dependent kinase (CDK) 4 and CDK6. The subset of DMP1 binding sites containing the GGA core can bind Ets family transcription factors Ets-1 and Ets-2. Screening of a series of natural promoters revealed that the CD13/aminopeptidase N (APN; EC 3.4.11.2) promoter could bind and be activated by DMP1. Activation of CD13/APN required both the intact DNA binding and transactivation domains of DMP1 and was inhibited by D-type cyclins, but not by cyclins A, B, C, or H, in a CDK-independent manner. CD13/APN is transactivated by a cooperative interaction between c-Myb bound to its cognate site and Ets-1 tethered to one of three GGA core-containing sites located 30-50 base pairs downstream. DMP1 binds to one of the Ets binding sites (designated Ets C) and synergizes with c-Myb in activating CD13/APN expression. Analysis of nuclear lysates from KG1a early myeloid cells using an oligonucleotide probe containing only the DMP1/Ets C binding site indicated that endogenous DMP1 and a putative Ets family member bind this element in vivo. DMP1-DNA complexes were significantly more stable than those containing the Ets factor. These data indicate that two different Myb family proteins collaborate in regulating APN gene expression and point to a role for DMP1 in normal myeloid cell development.