Methicillin-resistant Staphylococcus aureus strains with decreased vancomycin susceptibility have been isolated from patients in the United States and Japan. The impact of decreased vancomycin susceptibility on the drug’s pharmacodynamic parameters has not been addressed. We studied the activity of vancomycin against three clinical strains of vancomycin intermediate-susceptible Staphylococcus aureus (VISA) under high- and low-inoculum conditions, with stationary- and logarithmic-growth-phase kill curves, and in postantibiotic effect (PAE) experiments. We also investigated the stability of the decreased vancomycin susceptibility by using population susceptibility profiles. The respective vancomycin microdilution MICs and MBCs for VISA strains HIP5836, 14379, and Mu50 were 8 and 8, 8 and 8, and 8 and 16 μg/ml. HIP5836 had the most homogeneous elevation of vancomycin MICs, because the MIC for nearly all bacteria in the inoculum was 8 μg/ml. The population MICs (defined as the lowest vancomycin concentration inhibiting 99.9% of growth) for the first serial passages of HIP5836, Mu50, and 14379 were 8, 4, and 2 μg/ml, respectively. After 10 passages, they decreased to 4, 2, and 1 μg/ml, respectively. The Mu50 population MIC increased to 12 μg/ml after five serial passages on vancomycin agar. In the low- and high-inoculum kill curves, time to 99.9% killing was significantly (P < 0.05) longer for both Mu50 and HIP5836 than that for 14379 and a control strain. However, colony counts at 24 h were similar to those of the vancomycin-sensitive strain for all VISA strains. The PAE (at 4× MIC) ranged from 1.3 h for 14379 to 2.0 h for HIP5836 and was similar to or greater than the PAE against the vancomycin-sensitive strain. In conclusion, we found that the decreased vancomycin susceptibility increased during persistent exposures to the drug and decreased upon removal of the selective pressure. The decreased vancomycin susceptibility decreased the rate of vancomycin killing, but did not affect the extent of killing or the PAE.