Staphylococcus aureus with intermediate glycopeptide susceptibility (glycopeptide-intermediate S. aureus [GISA]) has been isolated from patients with apparent therapy failures. We studied the killing activity of vancomycin over a range of simulated conventional doses (1 to 1.5 g every 12 h) against three of these GISA strains in an in vitro pharmacodynamic infection model. We also studied the activity of a new glycopeptide (LY333328) at a simulated dose of 3 mg/kg of body weight every 24 h or 5 mg/kg every 24 h, as well as the potential for vancomycin and gentamicin synergy against these GISA strains. Four doses of vancomycin with or without gentamicin or two doses of LY333328 were administered over the 48-h study period. The vancomycin and LY333328 MICs and minimal bactericidal concentrations (MBCs) for the three GISA strains (strains 14379, 992, and Mu50) were 8 and 8 µg/ml and 1 and 2 µg/ml, respectively, for GISA 14379, 6 and 6 µg/ml and 1 and 1 µg/ml, respectively, for GISA 992, and 8 and 12 µg/ml and 2 and 8 µg/ml, respectively, for GISA Mu50. Vancomycin and LY333328 MICs and MBCs were 0.75 and 1.0 µg/ml and 1 and 1 µg/ml, respectively for a vancomycin-susceptible comparator strain (methicillin-resistant S. aureus [MRSA] 494). The addition of albumin to the growth medium increased the LY333328 MICs and MBCs approximately 8- to 16-fold. Vancomycin was bacteriostatic against the three GISA strains at doses of 1, 1.125, and 1.25 g every 12 h. Vancomycin was bactericidal at the dose of 1.5 g every 12 h against all strains; bactericidal activity occurred against the GISA strains at 8- to 10-fold lower ratios of the peak concentration to the MIC and the area under the concentration-time curve from time zero to 24 h (AUC_0-24) to the MIC compared to those for the vancomycin-sensitive control strain. Overall, vancomycin activity was significantly correlated with the AUC_0-24 (R² = 0.79; P < 0.001) by multiple stepwise regression analyses. The addition of gentamicin did not significantly affect killing activity against any strain. LY333328 was bactericidal against GISA strains 14379 and 992 and against MRSA 494 only with the 5-mg/kg/day dose simulations. The higher dose of LY333328 also prevented regrowth over the 48-h experiments for all strains tested. Higher doses of vancomycin (1.5 g every 12 h) and LY333328 (5 mg/kg every 24 h) may represent potential treatment options for infections caused by GISA strains.