In a search for proteins differentially cross-linked to DNA by cisplatin or formaldehyde in normal breast epithelial and breast cancer cell lines, we identified peroxiredoxin 1 (PRDX1) as a protein preferentially cross-linked to DNA in estrogen receptor negative (ER-) MDA-MB-231 but not in estrogen receptor positive (ER+) MCF7 breast cancer cells. Indirect immunofluorescence microscopic analyses showed that PRDX1 was located in the cytoplasm and nucleus of normal and breast cancer cells, with nuclear PRDX1 associated with promyelocytic leukemia protein bodies. We demonstrated that PRDX1 association with the transcription factor nuclear factor-kappaB (NF-kappaB) in MDA-MB-231 but not in MCF7 cells contributed to PRDX1-selective recruitment to MDA-MB-231 genomic DNA. Furthermore, PRDX1 was associated with the cyclooxygenase (COX)-2 upstream promoter region at sites occupied by NF-kappaB in ER- but not in ER+ breast cancer cells. PRDX1 knockdown attenuated COX-2 expression by reducing NF-kappaB occupancy at its upstream promoter element in MDA-MB-231 but not in MCF7 cells. A phosphorylated form of PRDX1 was only present in ER- breast cancer cells. Because PRDX1 phosphorylation is known to inhibit its peroxidase activity and to promote PRDX1 oligomerization, we propose that PRDX1 acts as a chaperone to enhance the transactivation potential of NF-kappaB in ER- breast cancer cells.