Zeste is a Drosophila regulatory gene that is required for transvection at the bithorax complex. Here we find that purified zeste protein binds to multiple sites just 5' of the initiation site of Ubx RNA. Zeste protein purified from Drosophila cells or from E. coli expressing the zeste gene activates Ubx transcription in vitro. This activation is dependent on the presence of zeste protein binding sites, as it is not observed with a Ubx promoter lacking these sites or with an Adh promoter. These results suggest that transvection involves regulatory elements that act at the level of transcriptional initiation and may be mechanistically similar to activation of transcription by enhancer elements, except that transvection occurs across paired chromosomes. These findings are consistent with the hypothesis that zeste may play a more important role in the normal regulation of Ubx and its other target genes than current genetic evidence implies.