The stratified epithelia of the oral cavity are continually exposed to bacterial challenge that is initially resisted by innate epithelial factors and by the recruitment of neutrophils. Antimicrobial peptides from phagocytes and epithelia contribute to this antimicrobial barrier. Using antibodies and in situ hybridization, we explored antimicrobial peptide expression in the varied epithelia of the periodontium and in cultured gingival epithelial cells. In gingival tissue, mRNA for the beta-defensins, human beta-defensin 1 (hBD-1) and human beta-defensin 2 (hBD-2) was predominately localized in suprabasal stratified epithelium and the peptides were detected in upper epithelial layers consistent with the formation of the stratified epithelial barrier. In cultured epithelial cells, both hBD-1 and -2 peptides were detected only in differentiating, involucrin-positive epithelial cells, although hBD-2 required stimulation by proinflammatory mediators or bacterial products for expression. Beta-defensins were not detected in junctional epithelium (JE) that serves as the attachment to the tooth surface. In contrast, alpha-defensins and cathelicidin family member LL-37 were detected in polymorphonuclear neutrophils (PMNs) that migrate through the JE, a localization that persists during inflammation, when the JE and surrounding tissue are highly infiltrated with PMNs. Thus, the undifferentiated JE contains exogenously expressed alpha-defensins and LL-37, and the stratified epithelium contains endogenously expressed beta-defensins. These findings show that defensins and other antimicrobial peptides are localized in specific sites in the gingiva, are synthesized in different cell types, and are likely to serve different roles in various regions of the periodontium.