AbstractTransposon mutagenesis provides a fundamental tool for functional genomics. Here we present a non-species-specific, combined enhancer detection and binary expression system based on the transposable element piggyBac: For the different components of this insertional mutagenesis system, we used widely applicable transposons and distinguishable broad-range transformation markers, which should enable this system to be operational in nonmodel arthropods. In a pilot screen in Drosophila melanogaster, piggyBac mutator elements on the X chromosome were mobilized in males by a Hermes-based jumpstarter element providing piggyBac transposase activity under control of the α1-tubulin promoter. As primary reporters in the piggyBac mutator elements, we employed the heterologous transactivators GAL4Δ or tTA. To identify larval and adult enhancer detectors, strains carrying UASp-EYFP or TRE-EYFP as secondary reporter elements were used. Tissue-specific enhancer activities were readily observed in the GAL4Δ/UASp-based systems, but only rarely in the tTA/TRE system. Novel autosomal insertions were recovered with an average jumping rate of 80%. Of these novel insertions, 3.8% showed homozygous lethality, which was reversible by piggyBac excision. Insertions were found in both coding and noncoding regions of characterized genes and also in noncharacterized and non-P-targeted CG-number genes. This indicates that piggyBac will greatly facilitate the intended saturation mutagenesis in Drosophila.