Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia, a disease resulting in morbidity and mortality of pigs and accordingly economic losses within the swine industry. In order to construct a potential vaccine strain of A. pleuropneumoniae for control of this disease, the aroQ gene, required for the aromatic biosynthetic pathway, was targeted for inactivation. The resulting strain was tested for virulence within pigs. The aroQ gene and an adjacent gene, dapD, were cloned. A recombination cassette, for inactivation of aroQ, was constructed from these cloned genes by inserting an ampicillin resistance gene and this was transformed into A. pleuropneumoniae. Integration of this construct into the chromosomal location of aroQ and disruption of the aroQ/dapD gene arrangement was confirmed through PCR and Southern analysis. The resulting HS25 aroQ mutants were unable to grow in a chemically defined medium and following intratracheal delivery to pigs were only able to induce lung lesions when given at a level 10,000 times greater than that of the parent strain. Complementation with an in trans, functional, aroQ gene restored the ability of the mutant strain to grow in a chemically defined medium and virulence, when tested in pigs, confirming attenuation results from inactivation of aroQ. In conclusion, this work has constructed a defined mutant of A. pleuropneumoniae that is attenuated and may be safely delivered live to pigs.