The activity of ampicillin-sulbactam against -lactamase-producing Escherichia coli has been questioned. Therefore, in this study, the killing activity of ampicillin-sulbactam was investigated in an in vitro infection model which simulates human pharmacokinetics. One ampicillin-sensitive strain (E. coli ATCC 25922, ampicillin-sulbactam MIC = 4/2 µg/ml) and three ampicillin-resistant TEM-1-producing strains with various levels of ampicillin-sulbactam resistance (EC11, MIC = 4/2 µg/ml; TIM2, MIC = 12/6 µg/ml; and GB85, MIC > 128/64 µg/ml) were studied. The E. coli strains were exposed to ampicillin-sulbactam at a starting inoculum of 6 to 7 log₁₀ CFU/ml. Ampicillin-sulbactam was infused over 30 min to simulate doses of 3 and 1.5 g every 6 h for 24 h. The 3-g ampicillin-sulbactam dose was bactericidal against E. coli ATCC 25922, EC11, and TIM2. The 1.5-g dose displayed bactericidal activity against ATCC 25922 and EC11 similar to that of the higher dose but failed to kill TIM2 due to inadequate time above the MIC and increased MICs over 24 h. GB85 was highly resistant and grew similarly to controls. Despite an MIC at 10⁷ CFU/ml indicating resistance (20/10 µg/ml), TIM2 was killed by the 3-g dose of ampicillin-sulbactam. Current MIC breakpoints may not adequately portray the activity of ampicillin-sulbactam, considering both the activity in in vitro infection models and clinical data.