The authors demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines, each expressing an exogenous gene in distinct small subsets of cells in the adult brain. They inserted overlapping 3-kb DNA fragments from genes thought to have patterned expression into a defined genomic location using site-specific recombination. These fragments were tested for their ability to function as transcriptional enhancers with a synthetic core promoter. Analysis of 44 fragments from four genes showed that over 80% drove specific expression patterns in the brain, with an average of fewer than 100 cells expressing the reporter gene. The results suggest that the D. melanogaster genome contains over 50,000 enhancers, each driving distinct subsets of gene expression in different tissues and developmental stages. The authors expect these lines to be valuable tools for neuroanatomy and the study of neuronal circuits and information flow in the fly brain.The authors demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines, each expressing an exogenous gene in distinct small subsets of cells in the adult brain. They inserted overlapping 3-kb DNA fragments from genes thought to have patterned expression into a defined genomic location using site-specific recombination. These fragments were tested for their ability to function as transcriptional enhancers with a synthetic core promoter. Analysis of 44 fragments from four genes showed that over 80% drove specific expression patterns in the brain, with an average of fewer than 100 cells expressing the reporter gene. The results suggest that the D. melanogaster genome contains over 50,000 enhancers, each driving distinct subsets of gene expression in different tissues and developmental stages. The authors expect these lines to be valuable tools for neuroanatomy and the study of neuronal circuits and information flow in the fly brain.