Actinobacillus pleuropneumonia strains that secrete three different exotoxins (ApxI, ApxII, and ApxIII) have been implicated in the etiology of porcine pleuropneumonia. To understand the role of these toxins in the pathogenesis of this disease, we have previously reported the cloning of the hemolysin gene (apxII) (Chang et al., 1989a), which encodes a 110-kD polypeptide with hemolytic and cytotoxic activity. To clone the third toxin gene (apxIII), a new genomic library using A. pleuropneumoniae serotype 2 chromosomal DNA was constructed. A series of five overlapping recombinant phage clones carrying the gene (apxIII) for this 120-kD antigen were identified using a DNA probe containing sequences from the Pasteurella haemolytica lktBD genes. Sequence analysis of a region of the cloned DNA reveals four open reading frames encoding proteins with predicted masses of 20.4, 112.5, 80.3, and 54.7 kD. These genes, designated apxIIC, apxIIIA, apxIIIB, and apxIIID, respectively, are similar in sequence to the RTX (repeat of toxin) toxin family. The toxin produced by the cloned gene kills BL-3 cells and is not hemolytic in vitro.