This mouse monoclonal antibody was generated against a ΦX174 bacteriophage-derived synthetic DNA–RNA antigen and recognizes RNA-DNA hybrids of various lengths. Highlights: High specificity and affinity for DNA-RNA hybrids Useful in the detection of R-loops Does NOT cross-react with single-stranded DNA or double-stranded DNA Minor cross-reaction (~5-fold less) has been observed for AU-rich double-stranded RNA. High affinity binding shown for hybrids of 8, 10, 15, and 23 base pairs in length DNA-RNA hybrids are a natural occurrence within eukaryotic cells, with levels of these hybrids increasing at sites with high transcriptional activity, such as during transcription initiation, repression, and elongation. Because RNA-DNA hybrids influence genomic instability, the S9.6 antibody is a useful reagent to help study the consequences of R-loops and lesions formed by these hybrids during DNA replication or other cellular processes. In addition, the S9.6 antibody is effective in recognizing RNA-DNA hybridization for microarray studies. From the laboratory of Stephen H. Leppla, PhD, National Institute of Allergy and Infectious Diseases/NIH.