Some Staphylococcus aureus isolates have glycopeptide minimal inhibitory concentrations (MICs) in the susceptible range but have subpopulations that grow on >or=4 microg/mL vancomycin. Clinical laboratory methods for determining susceptibility have proven to be inadequate for detecting these strains. Among methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) clinical isolates, 149 (66.2%) of 225 and 17 (56.6%) of 30, respectively, grew on brain-heart infusion (BHI) medium containing 2 microg/mL vancomycin; 17 (7.5%) of the MRSA and 2 (6.6%) of the MSSA isolates grew on BHI screening plates containing 4 microg/mL vancomycin. One isolate grew on plates containing 6 microg/mL vancomycin. This isolate escaped detection by routine testing but had a vancomycin MIC of 6 microg/mL when tested in BHI medium. This isolate also had decreased Triton X-100-induced autolysis and killing when incubated in broth media containing vancomycin, properties accorded to glycopeptide-intermediate S. aureus isolates. These observations suggest that glycopeptide-intermediate-like S. aureus isolates are circulating undetected and that a continuum of decreased susceptibility exists in unselected isolates.