Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air-liquid interface in low calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. P. gingivalis invaded intracellularly and spread cell to cell. A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer. S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced IL-1β, TNF-α, IL-6 and IL-8 secretion; and F. nucleatum stimulated production of IL-1β and TNF-α. A. actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, while the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.