Transposon mutagenesis is a powerful technique for generating collections of insertion mutants for genetic studies. This paper describes how phage Mu DNA transposition complexes, transpososomes, can be exploited for gene delivery to efficiently introduce selectable markers to genomes of Gram-positive bacteria. Mu transpososomes were assembled in vitro with custom-designed mini-Mu transposons, concentrated, and electroporated into cells of three Gram-positive bacterial species: Staphylococcus aureus, Streptococcus pyogenes and Streptococcus suis. Within cells, the complexes reproduced an authentic DNA transposition reaction and integrated the delivered transposons into the bacterial genomes, yielding single-copy insertions. The integration efficiency among different species and strains of Gram-positive bacteria ranged from 1x10(1) to 2x10(4) c.f.u. (mug introduced transposon DNA)(-1). The strategy should be applicable to a variety of other Gram-positive species after initial optimization of certain key factors affecting transposon delivery, such as the preparation method of competent cells and physical parameters of electroporation. This study extends the scope of the Mu transpososome delivery-based genomic DNA integration strategy to Gram-positive bacteria. Thus, a straightforward generation of sizeable mutant banks is feasible for these bacteria, potentiating several types of genomic-level approaches for studies of a variety of important bacterial processes, such as pathogenicity.