Members of the ets gene family have been implicated in the pathogenesis of a variety of hematologic malignancies. The Ets1 proto-oncogene is a DNA-binding, sequence-specific transcriptional activator of several cellular and viral gene promoters, including the long terminal repeat (LTR) of the human T-lymphotropic virus type I (HTLV-I). Ets1 has the ability to transform both erythroid and myeloid progenitor cells and has been implicated in the formation of certain leukemias. In our studies to understand adult T-cell leukemia/lymphoma (ATL), we have previously shown that Ets1 cooperatively interacts with HTLV-I Tax1 to synergistically transactivate the HTLV-I LTR and cellular promoters. We hypothesize that Ets1’s transcriptional effects on viral and cellular gene expression contributes to the development and manifestations of ATL. The interaction between Ets1 and Tax1 raises the possibility that Ets1 plays a role in the transformation of HTLV-I-infected T-lymphocytes and in the clinical manifestations of ATL. However, little is known about how Ets1 regulates gene expression and participates in transformation events. Electrophoretic mobility shift assays demonstrate that although Ets1 can bind to specific DNA elements, binding to the HTLV-I LTR and other promoters is not necessary for Ets1’s transcriptional activity when Tax1 is present. Deletion mutagenesis of Ets1 identifies that the amino terminal, central and distal carboxy terminal domains of Ets1 individually increase transcription from the HTLV-I LTR. The DNA-recognition domain-containing proximal carboxy terminal region of Ets1 inhibits HTLV-I LTR transcription. Only wild type Ets1 and a protein consisting of the entire carboxy terminus bind to the HTLV-I LTR. Transcriptional activity of truncated Ets1 proteins is not dependent on their LTR-binding ability. The synergistic Ets1/Tax1 interaction appears to require the involvement of additional cellular proteins, including the cAMP response element binding protein (CREB). In transient transfections of CREB-deficient cells, functional CREB cooperatively interacts with Ets1 to transactivate the HTLV-I LTR. In the presence of CREB, Tax1 has an additive effect on Ets1 transactivation. CREB appears to interact with specific Ets1 domains in mediating transcriptional activity and may involve an NF-kB pathway. SP1 transactivation of the HTLV-I LTR is independent of Ets1 or Tax1. Transient transfection of SP1-deficient cells reveals that SP1 transactivation of the HTLV-I LTR is independent of Ets1 or Tax1. Ets1 has no transcriptional activity on the HTLV-I LTR in the absence of SP1. These results demonstrate that: (1) Ets1 contains multiple functional domains that likely interact in mediating transcription from the HTLV-I LTR and which are not dependent on DNA binding; (2) Ets1 requires the presence of specific cellular and/or viral proteins to mediate its activities; and (3) HTLV-I Tax1, CREB, possibly NF-kB, and perhaps other cellular proteins mediate Ets1’s transcriptional activity. Further elucidation of Ets1 interactions with cellular and viral proteins is important for understanding Ets1’s role in cellular and disease processes.