In Staphylococcus aureus infection hemolysis caused by the extracellular protein alpha-toxin encoded by hla is thought to contribute significantly to its multifactorial virulence. In vitro, subinhibitory concentrations of beta-lactam antibiotics and fluoroquinolones increase the levels of hla and alpha-toxin expression, whereas aminoglycosides decrease the levels of hla and alpha-toxin expression. In the present study we investigated the effects of subinhibitory concentrations of amoxicillin, gentamicin, and moxifloxacin on hla and alpha-toxin expression and total hemolysis of S. aureus strain 8325-4, a high-level alpha-toxin producer, and its alpha-toxin-negative mutant, DU 1090, in vitro and in a rat model of chronic S. aureus infection. The levels of expression of hla and alpha-toxin and total hemolysis did not differ significantly when amoxicillin, gentamicin, or moxifloxacin was added to cultures of S. aureus strain 8325-4. In vivo, strain 8325-4 induced a significantly increased level of hemolysis in infected pouches compared to that in uninfected control pouches, but the hemolysis was reduced to control levels by treatment with doses of amoxicillin, gentamicin, or moxifloxacin that reduced bacterial numbers by 2 orders of magnitude. Additionally, the effects of subinhibitory concentrations of the three antibiotics on total hemolysis of four methicillin-resistant S. aureus and three methicillin-sensitive S. aureus (MSSA) clinical isolates were assessed in vitro. A significant increase in total hemolysis was observed for only one MSSA strain when it was treated with amoxicillin but not when it was treated with moxifloxacin or gentamicin. When purified alpha-toxin was incubated with purified human neutrophil elastase, alpha-toxin was cleaved nearly completely. The results suggest that the penicillin-induced increases in S. aureus alpha-toxin expression are strain dependent, that reduction of bacterial numbers in vivo counteracts this phenomenon effectively, and finally, that in localized S. aureus infections alpha-toxin activity is controlled by neutrophil elastase.