The role in virulence of Actinobacillus pleuropneumoniae urease activity was investigated. A urease-negative mutant was isolated following transposon mutagenesis with a mini-Tn10 derivative. Both the parent strain and the urease-negative mutant exhibited identical LD 5 0 values in a murine infection model. Pig challenge confirmed that the urease-negative mutant was fully virulent, since experimental inoculation with 5 10 7 colony forming units resulted in an acute disease indistinguishable from that produced by the wild-type strain at the same dose. Our results demonstrate that urease activity is not required for the development of acute pleuropneumonia.