Three benzylpenicillin-resistant, clinical isolates of Enterococcus faecium (MIC values 16-64 micrograms ml-1) contained six penicillin-binding proteins (PBPs), of which PBP5 was the most abundant and had the lowest affinity for the antibiotic. Four benzylpenicillin-susceptible strains (MIC values 0.031-0.5 microgram ml-1) were obtained as spontaneous derivatives from these above organisms. There were significant decreases in the amounts of PBP5 in each of the derivatives, with the concomitant appearance of a new, higher affinity PBP (5*) in three strains. Increased amounts of PBP5, with no changes in PBP5*, were found in several mutants with intermediate-level benzylpenicillin-resistance (MIC values 1-8 micrograms ml-1) selected from two of the susceptible strains. Examination of 18 other clinical isolates, with a wide range of susceptibilities to benzylpenicillin (MIC values 0.062-128 micrograms ml-1), showed that PBP5* was present in 13 strains, and PBP5 in all of them, but in differing amounts. The results concerning the relative amounts and relative affinities of PBPs 5* and 5 allowed the categorization of the various strains into six groups, within which organisms had somewhat similar susceptibilities to benzylpenicillin.