We have used a transient expression assay employing Drosophila tissue culture cells to study the potential of several Drosophila homeobox proteins to function as transcriptional regulators. A 96 bp fragment from the promoter region of the segment polarity gene engrailed, previously shown to contain five copies of a 10 bp consensus binding site for these proteins, enhanced transcription in the presence, but not the absence, of several different homeobox protein expression vectors. It is interesting that cotransfection with combinations of expression vectors encoding the homeobox proteins fushi tarazu, paired, and/or zen resulted in substantial synergistic increases in expression. In contrast, the products of the even-skipped and engrailed genes were found to repress, or quench, the activation induced by the other proteins. We discuss the implications of these results with respect to the role of homeobox genes in the control of embryonic development, and propose a "multi-switch" model whereby the activity of a target gene depends on the interactions of different homeobox proteins with multiple copies of a common binding site.