The involvement of the RTX haemolysins (ApxI and ApxII) of the swine pathogen Actinobacillus pleuropneumoniae in virulence was investigated using haemolysin-deficient mutants constructed by a mini-Tn10 mutagenesis procedure. Two types of haemolysin mutant with single insertions of the transposon were obtained from a serotype 1 strain producing both ApxI and ApxII. One presented a complete loss of haemolytic activity because of the absence of ApxI and ApxII production. The other displayed weaker haemolysis than the wild type and produced only ApxII. The chromosomal regions flanking mini-Tn10 were cloned and sequenced. In the non-haemolytic mutant, the transposon had inserted in apxIB, a gene involved in the exportation of ApxI and ApxII toxins. The weakly haemolytic mutant resulted from the disruption of the structural gene for ApxI. Both mutations in the apxI operon were associated with a significant loss of virulence for mice and pigs, demonstrating that haemolysins are involved in A. pleuropneumoniae pathogenicity. The non-haemolytic mutant was apathogenic and the weakly haemolytic mutant retained some virulence for pigs, suggesting that both ApxI and ApxII are needed for full virulence.