A new staphylococcal composite transposon, designated Tn5405, carrying the genes aphA-3 and aadE, which encode resistance to aminoglycosides, was partially characterized. The transposon is 12 kb long and is flanked by inverted repeated sequences displaying the characteristic features of an insertion sequence, named IS1182. This insertion sequence is 1864 bp long and has 23/33-bp imperfect inverted repeats at its ends. One of the IS1182 copies delimiting Tn5405 contains a copy of IS1181 flanked by 8-bp direct repeats. Tn5405 was found in the chromosome of MRSA clinical isolate BM3121, within a Tn552-related transposon, Tn5404. Tn5404 was previously characterized following its transposition onto a beta-lactamase plasmid harbored by BM3121. Two forms of the recombinant beta-lactamase-encoding plasmid generated by the inversion of Tn5405 within Tn5404 were detected. IS1182 was not detected in the DNA of 4 of the 17 tested MRSA isolates containing aphA-3 and resistant to streptomycin. Thus, aphA-3 and aadE genes are not disseminated only by Tn5405 or related transposons delimited by IS1182.