[en] Two plasmid-derived NcoI DNA fragments of 14 and 4.5 kb, respectively, have been isolated from the multidrug-resistant strain Enterococcus hirae S185R and analyzed. The 14-kb fragment contains two inverted (L and R) IS1216 insertion modules of the ISS1 family. These modules define a Tn5466 transposon-like structure that contains one copy of the methylase-encoding ermAM conferring erythromycin resistance and one copy of the adenylyl-transferase-encoding aadE conferring streptomycin resistance. Immediately on the left side of IS1216L there occurs a copy of pbp3r encoding the low-affinity penicillin-binding protein (PBP) PBP3r, itself preceded by a psr-like gene (psr3r) that controls the synthesis of PBP3r. ermAM, aadE, and the transposase gene (tnp) of IS1216R have the same polarities, and these are opposite those of psr3r, pbp3r, and the tnp gene of IS1216L. The 4.5-kb fragment is a copy of the 4.5-kb sequence at the 5' end of the 14-kb fragment, although it is not a restriction product of the 14-kb fragment. It contains three genes with the same polarity: psr3r, pbp3r, and tnp in an IS1216 element. Because of the very high degree of identity (99%) with the chromosomal psrfm and pbp5fm genes of Enterococcus faecium D63R, it is proposed that both the psr3r and pbp3r genes were transferred from an E.faecium strain and inserted in a plasmid of E. hirae. E. hirae is the first known bacterial species in which a low-affinity PBP-encoding gene has been found to be plasmid borne.