Forty-two clinical isolates belonging to ten species of staphylococci were studied for their ability to develop single-step resistance, in vitro, to glycopeptide antibiotics. Selection was attempted through separate exposure to four glycopeptides (vancomycin, teicoplanin, and two investigational semisynthetic derivatives of the latter, TD-A3 and CTA-A1) on agar containing 10 mg/l of the test drug. No survivors from any test strain were recovered after exposure to TD-A3 or CTA-A1. After exposure to vancomycin or teicoplanin, surviving clones were only recovered from strains of three species, Staphylococcus aureus, S. epidermidis, and S. haemolyticus. Emergence of resistant clones was easier to observe from strains of S. haemolyticus exposed to teicoplanin. When tested for susceptibility, many survivors exhibited vancomycin and teicoplanin MICs below the drug concentration used for in-vitro selection, probably due to an inoculum effect in the plating procedure. In particular, the vancomycin MICs did not exceed 8 mg/l for S. aureus and S. epidermidis clones, and reached 16 mg/l for some clones from a S. haemolyticus strain. Teicoplanin MICs did not exceed 8 mg/l for S. aureus clones, but reached 64 mg/l for some clones of S. epidermidis, and were particularly high (64 to greater than or equal to 128 mg/l) for most clones of S. haemolyticus. In contrast, against all clones selected from all three species, the MICs of TD-A3 and CTA-A1 did not exceed 2 and 4 mg/l, respectively. Morphological investigations indicated that the colonies of a highly resistant S. haemolyticus clone were smaller and more butyraceous in consistency than those of the parent strain. In transmission and scanning electron microscopy studies, this same S. haemolyticus clone showed a more irregular cell wall than the parent strain.