We have identified a cellular protein, RBP-2N, a presumed recombinase, as a repressor of transcription. Inhibition of transcription by RBP-2N was dependent on its DNA recognition site and was demonstrated in vitro and in vivo. This repression appears to be general, as transcription mediated by SP1 and Gal4/VP16 was inhibited by RBP-2N. The protein was purified to near homogeneity from human cells on the basis of its binding to a site present in the promoter of the adenovirus pIX gene. The DNA recognition sequence is 5′-TGGGAAAGAA, which is markedly different from the recombination signal sequence originally identified as the target site for this protein. The sequence of the purified protein is 97% identical with that published for the mouse RBP-2N protein. The reported homolog in Drosophila is Suppressor of Hairless. RBP-2N binding sites are present in a number of cellular and viral promoters, so RBP-2N may have a general role in transcriptional repression.