Prostate cancer cell lines were examined for proteins that partnered with the transcription factor C/EBP by use of a pull-down assay with S-tagged C/EBP combined with MALDI-TOF-MS analysis. Ku70, Ku80, and poly (ADP-ribose) polymerase-1(PARP-1) were identified as proteins that associated with C/EBP. The physical interaction of C/EBP(alpha) with these partner proteins was further demonstrated by GST-pull-downs using purified protein expressed in E. coli. The strongest binding was between C/EBP and PARP-1. Immunoprecipitation of C/EBPexpressed in prostate cancer cells co-precipitated Ku70, Ku80, and PARP-1. Deletion analysis of C/EBP indicated that the C-terminus of C/EBP was essential for the interaction of C/EBP with Ku70, Ku80, and PARP-1. Functional analysis of the interaction between C/EBP and the Ku proteins as well as PARP-1 showed that cells exhibiting these interactions had increased radiation sensitivity and decreased ability to repair double strand DNA breaks. Deficient DNA repair was dependent on the prostate cancer cell line tested, suggesting a complex process. We conclude that the association of C/EBP with Ku proteins and PARP-1 raises the likelihood that prostate cancer cells may be more sensitive to DNA damaging agents and may be important in the design of new prostate cancer therapies.