Among the twelve different serotypes of Actinobacillus pleuropneumoniae, the causative agent of swine pleuropneumonia, a strongly active hemolysin I (HlyI) is produced by serotypes which are particularly virulent, and less active hemolysin II (HlyII) is produced by all serotypes except type 10. In the serotypes 1, 5a, 5b, 9, 10 and 11, which produce HlyI, the hemolysin (hly) operon consists of a structural hlyIA gene, encoding pre-HlyI, an activator gene, hlyIC, necessary for the activation of pre-Hly to active Hly, and two genes, hlyIB and hlyID, involved in Hly secretion. These genes are clustered in the order, hlyICABD. This is characteristic to RTX toxin (repeats in the structural toxin) operons. The HlyII operons in all serotypes producing HlyII consist only of the pre-HlyII-encoding gene, appA, and its activator gene, appC. The serotypes, which produce HlyII, but not HlyI, contain a truncated HlyI operon, with the promoter, hlyIB and hlyID, and a small segment of the C terminus of hlyIA. This partial HlyI operon might have been formed by deletion of hlyIC and most of hlyIA. In serotype 3, which produces HlyII, but no HlyI, and which releases only minute amounts of this Hly into the growth medium, none of the hlyI genes and consequently no Hly secretion genes were found. The above results postulate that HlyII is secreted via the products of hlyIB and hlyID, and explain the low amount of HlyII secreted by serotype 3. Cloning and analysis of the structural genes encoding pre-HlyI and pre-HlyII among the different serotypes revealed differences in the hlyIA genes which are highly similar in the serologically related serotypes 1, 9 and 11, and differ from the serotypes, 5a, 5b and 10. The hlyIIA genes, in contrast, seem to be conserved in all serotypes.