Mating experiments have shown that high-level resistance (minimal inhibitory concentration greater than 2,000 microgram/ml) to streptomycin and kanamycin, and resistance to penicillin-streptomycin and penicillin-kanamycin synergism are transferable by conjugation from resistant clinical isolates of enterococci to a sensitive recipient strain. Cesium chloride-ethidium bromide ultracentrifugation revealed a satellite (plasmid) band in resistant clinical isolates and the transconjugant strains but not in the sensitive recipient. Examination of these satellite bands by agarose gel electrophoresis and electron microscopy demonstrated a common plasmid with a weight of 45 megadaltons. Novobiocin treatment of a resistant clinical isolate produced simultaneous loss of high-level resistance to streptomycin and kanamycin, and of resistance to penicillin-aminoglycoside synergism. These results suggest that (a) high-level resistance to streptomycin and kanamycin among some clinical isolates of enterococci is associated with a 45 megadalton plasmid, and (b) the same plasmid is also responsible for the resistance to penicillin-aminoglycoside synergism observed in these strains.