Background: Horizontal gene transfer (HGT) is a process by which bacteria acquire genes from organisms of distant taxa. HGT is now recognized as a major driving force in the evolution of bacterial pathogens. Through this process, bacteria may accumulate blocks of DNA such as genomic islands (GEIs) that encode fitness or virulence factors. The periodontal pathogen A. actinomycetemcomitans has been known to exhibit variable virulence potential. It is postulated that GEIs may play a role in modifying the virulence potential of A. actinomycetemcomitans. This study was initiated to identify and determine the distribution of GEIs in A. actinomycetemcomitans. Methods: Forty-seven A. actinomycetemcomitans strains of serotypes a through f were examined. Strain-specific variant DNA in the genomes of A. actinomycetemcomitans was identified by polymerase chain reaction (PCR) genomic mapping and sequenced to identify GEIs. The distribution of the GEIs among test strains of A. actinomycetemcomitans was determined by PCR analysis and Southern hybridization assays. Results: An ∼22 kb GEI of A. actinomycetemcomitans, designated AAI-1, was identified in five serotype b strains. The AAI-1 exhibits low %G+C and encodes proteins of phage, restriction modification systems, mobile elements, and other hypothetical proteins of unknown functions. The insertion of AAI-1 was found to cause truncation of A. actinomycetemcomitans genes at the insertion site. Conclusions: Some A. actinomycetemcomitans strains may harbor GEIs, which were acquired via HGT by the bacteria. The GEIs may increase the gene repertoire of A. actinomycetemcomitans. However, the insertion of the GEIs in A. actinomycetemcomitans may also cause truncation and inactivation of resident genes at the insertion sites. The virulence significance of such gain and loss of genes in A. actinomycetemcomitans remains to be determined.