Clinical isolates of Streptococcus faecium are characteristically resistant to beta-lactam antibiotics. Two strains, selected for hypersusceptibility to penicillin, were derived from normally resistant isolates treated with novobiocin. These strains were also found to be hypersusceptible to other beta-lactams. Differences in beta-lactam susceptibility between the original isolates and the hypersusceptible strains could not be attributed to alterations in penicillin-binding protein affinities, and no evidence of a relative permeability barrier was found in the resistant strains. Isolated cell membranes prepared from resistant strains were found to possess two protein bands which were absent or greatly diminished in the membranes of susceptible strains. Hypersusceptibility to beta-lactam antibiotics in these strains may be due to the absence or alteration of one or more cell membrane proteins distinct from the penicillin-binding proteins of these organisms.