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 Spl-deficient background and have been used in a complementation assay to identify functional domains of human transcription factor Spl. The SV40 early promoter, which contains six Spl binding sites (GC boxes), is induced up to 500-fold in Drosophila cells by the expression of Spl, whereas promoters with fewer sites are activated less efficiently. Analysis of Spl 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 sequencespecific transcription factors, such as the Drosophila zesfe 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.