We have used indirect immunofluorescence of polytene chromosomes to examine the chromatin distribution of a 52-kD Drosophila protein designated B52. B52 is localized to transcriptionally active loci and, at the highly decondensed heat shock loci, can be seen to bracket the RNA polymerase II fluorescence signals symmetrically. We have also examined the distribution of B52 on nonpolytene chromosomes in Drosophila cell cultures with an in vivo UV cross-linking method and find that, here too, B52 is associated with boundaries of transcriptionally active chromatin. The predicted primary amino acid sequence of B52 reveals two regions with similarities to a number of other proteins known to interact with nucleic acids.