We have previously reported that the buccal mucosa can support delayed type hypersensitivity (DTH) reactions to contact sensitizers. In the present study, we show that cells isolated from the buccal epithelium are able to present soluble exogenous antigens to specific T cells. Single cell suspensions obtained by enzymatic dispersion of buccal epithelial sheets could present the native protein antigen hen-egg lysozyme (HEL) to the I-Ak-restricted CD4+ T-cell hybridoma specific for a.a 46–61 on HEL. T-cell activation resulted in interleukin-2 (IL-2) production which could be inhibited by anti-major histocompatibility complex (MHC) class-II antibodies of pertinent specificity. Immunohistochemical staining of whole buccal epithelial sheets revealed that all MHC II positive cells had a dendritic morphology and expressed ATPase activity, indicating that these cells represent a major antigen-presenting cell (APC) population in this tissue. Furthermore, single cell suspensions isolated from buccal epithelium (BEC) after local in vivo administration of either a native soluble protein, a synthetic dodecapeptide, or a contact sensitizer were able to activate antigen-specific T cells ex vivo. Kinetic analyses indicated that maximal APC activity in the oral epithelium occurred within 1 hr after local antigen administration, and had essentially vanished after 24 hr. Conversely, APC activity was undetectable in draining cervico-mandibular lymph node cell suspensions recovered 1 hr after local antigen injection but became manifest after 3–24 hr. These observations suggest that dendritic cells can acquire antigens in the buccal epithelium and migrate to draining lymph nodes where they present processed antigen to MHC class II-restricted T cells. This APC population may thus be a critical element in the initiation of Th1-driven DTH responses in the oral mucosa.