A 426 bp fragment of infB, a housekeeping gene that encodes translation initiation factor 2, was sequenced from 59 clinical isolates and type strains of Actinobacillus species and sequences were compared. Partial sequences of 16S rRNA genes were also obtained. By comparing infB sequences, Actinobacillus pleuropneumoniae, Actinobacillus equuli, Actinobacillus suis, Actinobacillus ureae, Actinobacillus arthritidis, Actinobacillus hominis and two unnamed genomospecies showed more than 85 % similarity to the type strain of the type species of the genus, Actinobacillus lignieresii. The taxonomic position of Actinobacillus capsulatus was unresolved; this species is more remotely related to A. lignieresii. The two species A. lignieresii and A. pleuropneumoniae could not be clearly separated by infB sequence analysis. The phylogeny of the genus Actinobacillus based on infB analysis was essentially congruent with relationships inferred from 16S rRNA sequence comparisons and DNA hybridization studies. Discrepancies were encountered with single strains or taxa at the periphery of the genus. Greater intraspecies variation was observed with infB sequences than with 16S rRNA gene sequences, with notable exceptions. The apparent subdivision of some species by 16S rRNA analysis was most likely caused by RNA operon heterogeneity.