Replication protein A (RPA) is a single-strand DNA-binding protein with essential roles in DNA replication, recombination, and repair. It is necessary for the formation of the preincision complex that is required for proper incision of damaged DNA nucleotides during DNA repair. We have previously identified small molecule inhibitors (SMI) with the ability to disrupt RPA-binding activity to ssDNA. Further characterization of these RPA inhibitors was done using both lung and ovarian cancer cell lines. Lung cancer cell lines showed increased apoptotic cell death following treatment with the SMI MCI13E, with IC50 values of approximately 5 mmol/L. The ovarian cancer cell line A2780 and the p53-null lung cancer cell line H1299 were particularly sensitive to MCI13E treatment, with IC50 values less than 3 mmol/L. Furthermore, a cell-cycle effect was observed in lung cancer cell lines that resulted in a lengthening of either G1 or S-phases of the cell cycle following single-agent treatment. Sequential treatment with MCI13E and cisplatin resulted in synergism. Overall, these data suggest that decreasing DNA-binding activity of RPA via a SMI may disrupt the role of RPA in cell-cycle regulation. Thus, SMIs of RPA hold the potential to be used as single-agent chemotherapeutics or in combinationwith current chemotherapeutic regimens to increase efficacy.Mol Cancer Ther; 10(10); 1–11. 2011 AACR.