We describe a second-generation deficiency kit for Drosophila melanogaster composed of molecularly mapped deletions on an isogenic background, covering 77% of the Release 5.1 genome. Using a previously reported collection of FRT-bearing P-element insertions, we have generated 655 new deletions and verified a set of 209 deletion-bearing fly stocks. In addition to deletions, we demonstrate how the P elements may also be used to generate a set of custom inversions and duplications, particularly useful for balancing difficult regions of the genome carrying haplo-insufficient loci. We describe a simple computational resource that facilitates selection of appropriate elements for generating custom deletions. Finally, we provide a computational resource that facilitates selection of other mapped FRT-bearing elements that, when combined with the DrosDel collection, can theoretically generate over half a million precisely mapped deletions.T HE availability of chromosomal deletion collections is of considerable benefit to the Drosophila research community for gene mapping, the phenotypic characterization of alleles, and genomewide genetic interaction screens. A core deficiency kit, composed of 270 genetically heterogeneous deletions covering 92% of the genome, has been built up over many years by the Bloomington Drosophila Stock Center (BDSC; http:/ / flystocks.bio.indiana.edu/Browse/df-dp/dfkit-info.htm). Continuing efforts by the Bloomington Center are currently focused on expanding genome coverage by recovering deletions in the vicinity of haplo-insufficient regions (K. Cook, personal communication). Despite the considerable utility of this collection, it does, by its very nature, suffer from a number of limitations. These include a heterogeneous genetic background, the presence of uncharacterized second-site mutations, and, for most deletions, molecularly undefined breakpoints. More recently, two groups have taken advantage of two key technologies: large collections of transposon insertions precisely mapped to the Drosophila genome sequence and site-specific recombination, to develop tools for producing custom chromosomal deletions in homogeneous genetic backgrounds that are mapped to the genome sequence with single-base-pair resolution (Parks et al. 2004;Ryder et al. 2004;Thibault et al. 2004).Sequence data from this article have been deposited with the EMBL/ GenBank data libraries under accession nos. AJ545047-AJ547612 and AJ622065-AJ622812. In both cases, the new deletion collections are generated using FLP-mediated recombination between pairs of transposon-borne FRT sites, a method originally developed in Drosophila by Golic and Golic (1996). In one case (Parks et al. 2004), a set of .29,000 P-element and piggyBac insertions (Thibault et al. 2004) were used to generate 519 deletions covering 56% of the euchromatic genome (the Exelixis collection). The high number of starting insertions used by this group allows fine-scale coverage of the genome with relatively small deletions; the average size of the exist...