The ability to visualize and manipulate cell fate and gene expression in specific cell populations has made gene expression systems valuable tools in developmental biology studies. Here, we describe a new system that uses the E. coli tryptophan repressor and its upstream activation sequence (TrpR/tUAS) to drive gene expression in stable zebrafish transgenic lines and in mammalian cells. We show that TrpR/tUAS transgenes are not silenced in subsequent generations of zebrafish, which is a major improvement over some of the existing systems, such as Gal4/gUAS and the Q-system. TrpR transcriptional activity can be tuned by mutations in its DNA-binding domain, or silenced by Gal80 when fused to the Gal4 activation domain. In cases in which more than one cell population needs to be manipulated, TrpR/tUAS can be used in combination with other, existing systems.