Abstract Using the yeast GAL4 transcription factor to control expression in Drosophila melanogaster has long been ineffective in female germ cells during oogenesis. Here, DeLuca and Spradling show that the expression problem of most Drosophila molecular tools... Controlling the expression of genes using a binary system involving the yeast GAL4 transcription factor has been a mainstay of Drosophila developmental genetics for nearly 30 years. However, most existing GAL4 expression constructs only function effectively in somatic cells, but not in germ cells during oogenesis, for unknown reasons. A special upstream activation sequence (UAS) promoter, UASp was created that does express during oogenesis, but the need to use different constructs for somatic and female germline cells has remained a significant technical limitation. Here, we show that the expression problem of UASt and many other Drosophila molecular tools in germline cells is caused by their core Hsp70 promoter sequences, which are targeted in female germ cells by Hsp70-directed Piwi-interacting RNAs (piRNAs) generated from endogenous Hsp70 gene sequences. In a genetic background lacking genomic Hsp70 genes and associated piRNAs, UASt-based constructs function effectively during oogenesis. By reducing Hsp70 sequences targeted by piRNAs, we created UASz, which functions better than UASp in the germline and like UASt in somatic cells.