Mos1 and other mariner/Tc1 transposons move horizontally during evolution, and when transplanted into heterologous species can transpose in organisms ranging from prokaryotes to protozoans and vertebrates. To further develop the Drosophila Mos1 mariner system as a genetic tool and to probe mechanisms affecting the regulation of transposition activity, we developed an in vitro system for Mos1 transposition using purified transposase and selectable Mos1 derivatives. Transposition frequencies of nearly 10-3/target DNA molecule were obtained, and insertions occurred at TA dinucleotides with little other sequence specificity. Mos1 elements containing only the 28 bp terminal inverted repeats were inactive in vitro,while elements containing a few additional internal bases were fully active, establishing the minimal cia-acting requirements for transposition. With increasing transposase the transposition frequency increased to a plateau value, in contrast to the predictions of the protein overexpression inhibition model and to that found recently with a reconstructed Himar1 transposase. This difference between the 'natural' Mos1 and 'reconstructed' Himar1 transposases suggests an evolutionary path for down-regulation of mariner transposition following its introduction into a naive population. The establishment of the cis and trans requirements for optimal mariner transposition in vitro provides key data for the creation of vectors for in vitro mutagenesis, and will facilitate the development of in vivo systems for mariner transposition.