A fourth type of RTX determinant was identified in Actinobacillus pleuropneumoniae and was designated apxlVA. When expressed in Escherichia coli, recombinant ApxlVA showed a weak haemolytic activity and cohaemolytic synergy with the sphingomyelinase (beta-toxin) of Staphylococcus aureus. These activities required the presence of an additional gene, ORF1, that is located immediately upstream of apxlVA. The apxlVA gene product could not be detected in A. pleuropneumoniae cultures grown under various conditions in vitro; however, pigs experimentally infected with A. pleuropneumoniae serotypes 1, 5 and 7 started to produce antibodies that reacted with recombinant ApxlVA 14 d post-infection, indicating that apxlVA is expressed in vivo. In addition, sera from pigs naturally and experimentally infected with any of the serotypes all reacted with recombinant ApxlVA. The apxlVA gene from the serotype 1 A. pleuropneumoniae type strain Shope 4074T encodes a protein with a predicted molecular mass of 202 kDa which has typical features of RTX proteins including hydrophobic domains in the N-terminal half and 24 glycine-rich nonapeptides in the C-terminal half that bind Ca2+. The glycine-rich nonapeptides are arranged in a modular structure and there is some variability in the number of modules in the ApxIVA proteins of different serotypes of A. pleuropneumoniae. The deduced amino acid sequences of the ApxlVA proteins have significant similarity with the Neisseria meningitidis iron-regulated RTX proteins FrpA and FrpC, and to a much lesser extent with other RTX proteins. The apxlVA gene could be detected in all A. pleuropneumoniae serotypes and seems to be species-specific. Although the precise role of this new RTX determinant in pathogenesis of porcine pleuropneumonia needs to be determined, apxlVA is the first in vivo induced toxin gene that has been described in A. pleuropneumoniae.