Ets-1 is the founding member of the Ets family of transcription factors, which are characterized by a well-conserved DNA-binding domain, called the ETS domain. It recognizes specific DNA elements, called Ets binding sites (EBS) that are present in the promoters of its target genes. This factor controls genes implied in various biological and pathological processes, such as development, haematopoiesis, angiogenesis, apoptosis and tumor invasion. In human, two isoforms of the Ets-1 protein were described: a majority isoform, the full-length Ets-1 p51, and Ets-1 p42 which results from an alternative splicing. A third isoform, Ets-1 p27, was recently discovered by our team. It is also generated by alternative splicing and acts as a dominant-negative towards Ets-1 p51-mediated transcriptional trans activation. Ets-1 regulates transcription of its target promoters via interactions with other nuclear proteins and transcription factors according to the cellular context. Moreover, it is a nuclear target of many signal transduction pathways. Identification of new proteins interacting with Ets-1 should permit to better understand molecular mechanisms involved in the regulation of its activity. For this purpose, we used an affinity purification strategy of Ets-1 partners using streptavidin pull-down. This approach requires recombinant biotinylated Ets-1 isoforms, produced using a prokaryotic expression system that we developed. Using MALDI-TOF mass spectrometry, several potential interaction partners were identified. Among those, we have determined the Poly (ADP-Ribose) Polymerase-1 (PARP-1) and the DNA-Dependent Protein Kinase (DNA-PK) complex, including its regulatory heterodimers, Ku70/Ku80 and its catalytic subunit, DNA-PKcs. The interaction between these proteins and endogenous Ets-1 were confirmed by co-immunoprecipitation in the cell. Previous studies have revealed that besides its role in DNA repair pathway, DNA-PK phosphorylates in vitro a number of transcription factors, such as p53, c-Fos, and c-Jun. Interestingly, in vitro phosphorylation assays revealed that DNA-PK was able to phosphorylate Ets-1. The role of PARP-1 on Ets-1-containing DNAPK complex is not yet well elucidated, but we suggest that it could stimulate DNA-PK kinase activity in Ets-1 phosphorylation. Thus, DNA-PK and PARP-1 by modulating Ets-1 phosphorylation might be involved in regulation of its transcriptional activity.