Expressing a gene in cells in which it is not normally active is a powerful way of determining its function. The GAL4 system allows the selective expression of any cloned gene in a wide variety of cell- and tissue-specific patterns in Drosophila. A promoter (or enhancer) directs expression of the yeast transcriptional activator GAL4 in a particular pattern, and GAL4 in turn directs transcription of the GAL4-responsive (UAS) target gene in an identical pattern. The system's key feature is that the GAL4 gene and UAS-target gene are initially separated into two distinct transgenic lines. In the GAL4 line, the activator protein is present, but has no target gene to activate. In the UAS-target gene line, the target gene is silent because the activator is absent. It is only when the GAL4 line is crossed to the UAS-target gene line that the target gene is turned on in the progeny. In this article we describe, in detail, how to generate and characterize GAL4 lines and how to prepare UAS-target gene lines. Vector maps are provided for pGaTB, P[GawB], and pP[UAST]. In addition, we consider the range of UAS-reporters currently available and review several new modifications of the GAL4 system.