DNA adenine methyltransferase (DAM) plays critical roles in diverse biological pathways in gram-negative bacteria, and specifically in regulating the expression of virulence genes in several organisms. Actinobacillus actinomycetemcomitans plays an important role in the pathogenesis of juvenile and adult periodontal disease, yet little is known about its mechanisms of gene regulation. DAM is shown here to directly or indirectly affect well-known A. actinomycetemcomitans virulence factors. A mutant A. actinomycetemcomitans strain lacking the dam gene was created by homologous recombination and shows normal growth phenotypes when grown exponentially. This mutant strain has four sixfold increased levels of extracellular leukotoxin, altered cellular levels of leukotoxin, and significant changes in bacterial invasion of KB oral epithelial cells. These results provide a basis for further characterization of regulatory mechanisms that control A. actinomycetemcomitans virulence.