Ten epidemiologically unrelated Staphylococcus aureus isolates with borderline levels of susceptibility to antistaphylococcal penicillinase-resistant penicillins (PRPs) were investigated together with appropriate S. aureus control strains. By a nitrocefin microplate assay, all borderline PRP-susceptible test strains were found to produce comparable amounts of beta-lactamase. Hydrolytic activity against another chromogenic substrate (PADAC) and against the PRPs was also demonstrated in membrane preparations from induced cells of 9 of the 10 borderline test strains. When bacterial membranes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two methicillin-inducible bands of about 32 and 31 kDa were detected, after Coomassie blue staining, in the membrane protein patterns of the same nine borderline test strains. By gel renaturation and zymographic detection of beta-lactamase activity, both methicillin-inducible membrane proteins were detected with nitrocefin as a substrate, whereas only one band (presumably the smaller protein) was detected with PADAC. With the remaining borderline test strain (a40), only the larger band was detected in the renatured gels with nitrocefin as a substrate. Plasmid DNA analysis revealed that the borderline susceptible test strains, again with the exception of a40, shared a 17.2-kb plasmid yielding four HindIII fragments of 7.0, 5.3, 3.5, and 1.4 kb. In Western blot (immunoblot) experiments using rabbit antiserum to penicillin-binding protein (PBP) 2a, test strain a40, which did not share a number of features characteristically associated with the other borderline test strains, was eventually shown to produce PBP 2a. Five other S. aureus strains, belonging to phage group 94/96, were found to display the borderline phenotype, including such distinguishing features as the membrane-associated PRP- and PADAC-hydrolyzing activity, the two methicillin-inducible membrane proteins, and the 17.2-kb plasmid. These results suggest that borderline susceptible S. aureus strains share more common features than reduced susceptibility to PRPs.