The conjugative shuttle transposon Tn1545 from Streptococcus pneumoniae confers resistance to kanamycin (aphA-3), erythromycin (ermAM), and tetracycline (tetM). The 25.3-kb element is self-transferable to various gram-positive bacterial genera where it transposes. Tn1545 is also capable of transposition, but not of conjugation, after cloningoff Escherichia coli. Analysis of the element by restriction endonucleases, molecular cloning, electron microscopy of heteroduplexes, DNA hybridization, and sequencing allowed us to establish a physical map of Tn1545, localize the resistance genes, determine their direction of transcription, and compare them with other characterized resistance determinants, and show that Tn1545 is not flanked by large terminal repeated sequences in opposite orientation.