We tested the combined activity of vancomycin and seven beta-lactam antibiotics against Staphylococcus aureus clinical strain Mu3, which displays heterogeneous resistance to vancomycin. When combined with vancomycin, four of the seven tested beta-lactams exhibited an additive effect at or near their MICs, while all showed an antagonistic effect at lower, sub-MIC levels. This study implicated the unpredictable nature of combination therapy of beta-lactams and vancomycin against S. aureus with reduced susceptibility to vancomycin.