Replication Protein A (RPA) is required for DNA recombination, repair and replication in all eukaryotes. RPA participation in these pathways is mediated by single-stranded DNA binding and protein interactions. We have identified a novel RPA protein interactor, Replication Protein Binding Trans-Activator (RBT1), in a yeast two-hybrid assay employing the second subunit of human RPA (RPA32) as bait. RBT1-RPA32 binding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation. Fluorescence microscopy indicates that over-expressed green fluorescence protein-tagged RBT1 is localized to the nucleus in vivo. RBT1 mRNA expression, determined by semi-quantitative RT-PCR, is significantly higher in cancer cell lines MCF-7, ZR-75, SaOS-2 and H661, compared to normal non-immortalized human mammary and bronchial epithelial cells. Further, yeast and mammalian one-hybrid analysis shows that RBT1 is a strong transcriptional co-activator. Interestingly, mammalian transactivation data are indicative of significant variance between cell lines; the GAL4-RBT1 fusion protein has significantly higher transcriptional activity in human cancer cells compared to human normal primary non-immortalized epithelial cells. NIH3T3 mouse fibroblasts stably overexpressing RBT1 meet several criteria of transformed cells, including lack of contact growth inhibition, growth of colonies in soft agar and tumor formation in nude mice; HCT116 colon carcinoma cells overexpressing RBT1 exhibit an enhanced growth rate in culture and form extremely aggressive tumors in vivo.