Prostate cancer cell lines were examined for proteins that partnered with the transcription factor C/EBPalpha by use of a pull-down assay with S-tagged C/EBPalpha combined with matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis. Ku70, Ku80, and poly(ADP-ribose) polymerase-1 (PARP-1) were identified as proteins that associated with C/EBPalpha. The physical interaction of C/EBPalpha with these partner proteins was further demonstrated by glutathione S-transferase (GST) pull-downs using purified protein expressed in Escherichia coli. The strongest binding was between C/EBPalpha and PARP-1. Immunoprecipitation of C/EBPalpha expressed in prostate cancer cells co-precipitated Ku70, Ku80, and PARP-1. Deletion analysis of C/EBPalpha indicated that the C terminus of C/EBPalpha was essential for the interaction of C/EBPalpha with Ku70, Ku80, and PARP-1. Functional analysis of the interaction between C/EBPalpha 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/EBPalpha with Ku proteins and PARP-1 raises the likelihood that C/EBPalpha-expressing prostate cancer cells may be more sensitive to DNA-damaging agents and may be important in the design of new prostate cancer therapies.