The highly organized structure of the Drosophila ovary makes it an ideal system for studying mechanisms of differential gene expression. Here we report the identification of a 171 bp sequence from the 5' end of the hsp26 gene that functions as a female germ-line-specific transcriptional regulator when linked in two copies to a basal promoter. The regulator is active only in nondividing cells of the germ line, i.e., only in nurse cells and oocytes. It is not active in any examined tissue or cell type outside of the female germ line. Copper nuclease footprinting studies show that the germ line regulator contains two binding sites for each of two different ovarian nuclear factors. Point mutations in the DNA target sites of either nuclear factor abolish in vitro binding and in vivo transcriptional activity, indicating that each factor is a positive activator of nurse cell/oocyte transcription. The two factors may represent different classes of activator proteins, since an increase in the copy number of one factor's DNA target site cannot compensate for a decrease in the copy number of the other factor's target site.