We have adopted Drosophila tissue culture cells as a host system for studying the structure and function of mammalian transcription factors. These cells provide an Sp1-deficient background and have been used in a complementation assay to identify functional domains of human transcription factor Sp1. The SV40 early promoter, which contains six Sp1 binding sites (GC boxes), is induced up to 500-fold in Drosophila cells by the expression of Sp1, whereas promoters with fewer sites are activated less efficiently. Analysis of Sp1 mutants reveals multiple distinct regions outside of the DNA binding domain that are responsible for mediating transcriptional activation. The two most active domains, which appear to be functionally redundant with one another, consist of an unusual structure with a very low charge density, but a strikingly high glutamine content. A number of other sequence-specific transcription factors, such as the Drosophila zeste protein and several homeodomain proteins, contain glutamine-rich stretches, and we propose that these glutamine-rich domains represent a novel structural motif for transcriptional activation.