The use of fluorescent protein tags has had a huge impact on cell biological studies in virtually every experimental system. Incorporation of coding sequence for fluorescent proteins such as green fluorescent protein (GFP) into genes at their endogenous chromosomal position is especially useful for generating GFP-fusion proteins that provide accurate cellular and subcellular expression data. We tested modifications of a transposon-based protein trap screening procedure in Drosophila to optimize the rate of recovering useful protein traps and their analysis. Transposons carrying the GFP-coding sequence flanked by splice acceptor and donor sequences were mobilized, and new insertions that resulted in production of GFP were captured using an automated embryo sorter. Individual stocks were established, GFP expression was analyzed during oogenesis, and insertion sites were determined by sequencing genomic DNA flanking the insertions. The resulting collection includes lines with protein traps in which GFP was spliced into mRNAs and embedded within endogenous proteins or enhancer traps in which GFP expression depended on splicing into transposon-derived RNA. We report a total of 335 genes associated with protein or enhancer traps and a web-accessible database for viewing molecular information and expression data for these genes. A S a model organism, perhaps the most important advantage of Drosophila is the extensive range of very sophisticated genetic tools available. Central among these are transposons, including P elements and piggyBac (PBac) elements, which can be used to create easily mapped insertion mutations and to analyze gene expression with a variety of reporter molecules. P elements are naturally occurring Drosophila transposable elements that were first modified to provide vectors for efficient DNA-mediated gene transfer in Drosophila (Rubin and Spradling 1982) and then to create collections of random, single-element insertions in the genome (Robertson et al. 1988;Cooley et al. 1989;Spradling et al. 1995Spradling et al. , 1999Bellen et al. 2004). Although very useful for mutagenesis, P elements also have limitations including preference for the 59 region of genes (O'Hare and Rubin 1983;Tsubota et al. 1985), bias toward particular sequence motifs (O'Hare and Rubin 1983), and ''hot spots'' that have been hit at a high frequency (Spradling et al. 1999).The more recent use of transposons based on the Lepidopteran PBac element has expanded the number of genes disrupted by single transposon insertions (Horn et al. 2003;Bellen et al. 2004;Bonin and Mann 2004;Thibault et al. 2004). First introduced into the Drosophila melanogaster germline by Handler and Harrell (1999), the PBac elements were shown to transpose and insert at TTAA sequences. Like the P element, PBac contains a single open reading frame encoding transposase and is bounded by short terminal inverted repeats. PBac elements have been demonstrated to insert into new genes that have not previously been hit using P-element techniques.Engineered tr...