The Polycomb group (PcG) of genes are required for maintenance of the repressed state of the homeotic genes in Drosophila. There are similarities between the PcG repression and mating-type silencing in yeast or heterochromatic position effect in Drosophila, which suggest that PcG repression may involve a highly compacted chromatin structure. To test for such a structure, heterologous DNA- binding proteins were used as probes for DNA accessibility in Drosophila embryos. Binding sites for the yeast transcriptional activator GAL4 and for bacteriophage T7 RNA polymerase were inserted into the bithorax (bx) regulatory region of the endogenous Ultrabithorax (Ubx) gene, which is regulated by the PcG. Ubiquitously expressed GAL4 protein directs transcription through its binding sites only in the posterior segments where the bx region is active. The block to GAL4 activation in the more anterior segments is dependent on Polycomb (Pc) function. In contrast, T7 RNA polymerase can transcribe from its target promoter in all segments of the embryo. Thus, Pc-mediated repression blocks activated polymerase II transcription, but does not simply exclude all proteins.