RI(dl)-2 is a cell-permeable pyrroloquinoxaline derivative th at selectively inhibits Rad51-mediated D-loop formation (IC50 = 11.1 μM) without affecting its ssDNA-binding activity (IC50 >100 μM). RI(dl)-2 is shown to inhibit DSBs-induced cellular homologous recombination (HR) activity (IC50 = 3.0 μM by HEK293-based DR-GFP reporter assay) by stabilizing nucleoprotein filaments in a nonfunctional state without affecting DNA damage-induced RAD51 nuclear loci formation or stimulating single-strand annealing (SSA) activity.