Transgenesis is a process of introducing foreign genetic material into the genomes of living organisms. One of the tools for transgenesis are the transposable elements (TEs), which include transposons. Transposons are naturally occurring sequences of DNA which are recognised, excised and inserted into a new location by a single enzyme – transposase. Here we show studies of the biophysical properties and activities of two highly related eukaryotic TEs of the mariner family: Mos1 from Drosophila mauritiana and Mboumar-9 (Mbo9) from Messor bouviery. Using biochemical and molecular methods we examined the properties of transposases in vitro and in vivo. Recombinant transposases were expressed in E.coli and purified using HPLC. Each protein’s activity was assayed for cleavage, integration and the whole transposition reaction. We used a modelling approach to predict the structure of the complex of Mbo9 transposase bound to the specific terminal sequences of the transposon, the paired end complex (PEC), based on the published crystal structure of Mos1 PEC. We have found that both transposases are elongated dimers in solution and that the first helix-turn-helix domain is involved in the protein dimerization. Moreover we show that mariner transposases cut one of the imperfect inverted repeats more efficiently than the other. The terminal nucleotide of the inverted repeat is important for integration of the transposon into a new target DNA, while having no effect at the stage of cleavage. Previously, neither Mos1 nor Mbo9 had been shown to have significant activity in mammalian cells. We have developed a new assay that allows chromosomal integration of the desired DNA sequence in vivo in bacterial, yeast and mammalian cells without the use of helper plasmids or mRNA injection. We found the optimal combination of inverted repeats for each of the transposons and have enhanced the transposition efficiency of Mbo9 by changing the sequence of its inverted repeat DNA. This study is a foundation for improving mariner TEs for transgenesis.