Activator/Dissociation (Ac/Ds) transposable elements from maize are widely used as insertional mutagenesis and gene isolation tools in plants and more recently also in medaka and zebrafish. They are particularly valuable for plant species that are transformation-recalcitrant and have long generation cycles or large genomes with low gene densities. Ac/Ds transposition frequencies vary widely, however, and in some species they are too low for large-scale mutagenesis. We discovered a hyperactive Ac transposase derivative, AcTPase 4x , that catalyzes in the yeast Saccharomyces cerevisiae 100-fold more frequent Ds excisions than the wild-type transposase, whereas the reintegration frequency of excised Ds elements is unchanged (57%). Comparable to the wild-type transposase in plants, AcTPase 4x catalyzes Ds insertion preferentially into coding regions and to genetically linked sites, but the mutant protein apparently has lost the weak bias of the wild-type protein for insertion sites with elevated guanine-cytosine content and nonrandom protein-DNA twist. AcTPase 4x exhibits hyperactivity also in Arabidopsis thaliana where it effects a more than sixfold increase in Ds excision relative to wild-type AcTPase and thus may be useful to facilitate Ac/Ds-based insertion mutagenesis approaches. DNA transposons are widely used in plants and animals as functional genomics tools and gene transfer vehicles. In plants, transposable elements are particularly valuable when large-scale T-DNA insertion mutagenesis is not feasible, e.g., for transformation-recalcitrant species or plants with long generation cycles. The success of transposon insertion mutagenesis strategies depends on high forward mutagenesis rates and a favorable distribution of novel insertions. As forward mutagenesis rates are frequently limited by transposase activity, attempts were made to find hyperactive transposase mutants. For some transposons, such mutants were fortuitously found; in other instances, systematic screening approaches and molecular evolution were successful (Goryshin and Reznikoff 1998;Beall et al. 2002;Baus et al. 2005;Keravala et al. 2006;Mates et al. 2009).The maize Activator/Dissociation (Ac/Ds) transposable elements have been widely used in plants for gene tagging and functional genomics approaches because they are active in numerous plant species, integrate preferentially into or near to coding regions, and frequently transpose to genetically linked sites, enabling local saturation mutagenesis approaches (reviewed in Kunze and Weil 2002). The successful introduction of Ac/Ds elements into yeast revealed that application of these elements is not restricted to plants (Weil and Kunze 2000). Their recent adoption as tools for transgenesis and the generation of gene trap lines in the teleost fishes zebrafish and medaka further emphasized the wide range functionality of Ac/Ds elements (Emelyanov et al. 2006;Boon Ng and Gong 2011;Froschauer et al. 2012).An impediment for a universal application is the variability of Ac/Ds transposition fr...