The scs and scs' elements were proposed to function as chromatin domain boundaries for the 87A7 heat shock locus in Drosophila melanogaster. Here we report the identification and characterization of SBP (scs binding protein), a component of the scs nucleoprotein complex. SBP binds specifically to a 24-bp region of scs in vitro and is associated with scs in vivo. Multiple copies of an oligonucleotide containing the SBP recognition sequence are capable of blocking enhancer-promoter interactions in transgene assays. Mutations in the oligonucleotide that disrupt SBP binding in vitro also eliminate enhancer-blocking activity in vivo. We show that SBP is encoded by the zeste-white 5 gene and that mutations in zeste-white 5 reduce the enhancer-blocking activity of the multimerized oligonucleotides.