Actinobacillus pleuropneumoniae is the causative agent of an economically significant form of porcine pleuropneumonia. This bacterium produces four distinct RTX toxins (ApxI-ApxIV) that play a key role in its pathogenesis, but further characterization of these hemolytic toxins is needed to identify the environmental signals and genes that affect their production during infection. In this report, we examined the effect of oxygen limitation on the production of ApxI and ApxII, the two RTX toxins that are produced by all highly virulent strains in North America. Batch cultures of ApxI- and ApxII-producing strains were grown in heart infusion broth supplemented with NAD, and samples were prepared throughout the growth curve. We compared batch cultures with normal oxygen levels to those that were maintained in an oxygen-depleted state. ApxI and ApxII hemolytic activity were nearly identical under both growth conditions. The level of toxin activity was confirmed by examination of extracellular ApxI concentrations and apxII mRNA levels. In addition, toxin activity examined on blood agar plates grown aerobically or anaerobically showed no significant difference. These results have important implications for the disease state where high-density growth is likely to result in local anaerobic or microaerophilic environments.