Vaccination against Actinobacillus pleuropneumoniae is hampered by the lack of vaccines inducing reliable cross-serotype protection. In contrast, pigs surviving natural infection are at least partially protected from clinical symptoms upon reinfection with any serotype. Thus, we set out to construct an attenuated A. pleuropneumoniae live vaccine allowing the differentiation of vaccinated from infected animals (the DIVA concept) by successively deleting virulence-associated genes. Based on an A. pleuropneumoniae serotype 2 prototype live negative marker vaccine (W. Tonpitak, N. Baltes, I. Hennig-Pauka, and G.-F. Gerlach, Infect. Immun. 70:7120-7125, 2002), genes encoding three enzymes involved in anaerobic respiration and the ferric uptake regulator Fur were deleted, resulting in a highly attenuated sixfold mutant; this mutant was still able to colonize the lower respiratory tract and induced a detectable immune response. Upon a single aerosol application, this mutant provided significant protection from clinical symptoms upon heterologous infection with an antigenically distinct A. pleuropneumoniae serotype 9 challenge strain and allowed the serological discrimination between infected and vaccinated groups.