Allergic contact dermatitis is a T-cell-mediated inflammation, induced by contact with sensitizers and occurring through the release of epidermal cytokines and the activation of epidermal Langerhans cells (LCs). The aim of this study was to analyse early events of LC activation induced either by contact allergens or by irritants devoid of any contact allergenic properties, in order to obtain an in vitro method to discriminate between these two groups of molecules. Various contact sensitizers and irritants were studied for their effects on the endocytosis of major histocompatibility complex class II (MHC-II) molecules by freshly-isolated human epidermal LCs. As observed by flow cytometry, a spontaneous decrease in the surface expression of MHC-II (HLA-DR) molecules, linked to spontaneous internalization of the MHC-II molecules by LCs, was obtained by moving freshly-isolated LCs from 4 °C to 37 °C. Pre-incubation of LCs with either sensitizers or irritants increased the spontaneous internalization of HLA-DR molecules with a similar magnitude, but no clear discrimination between sensitizer and irritant effects was obtained by flow cytometry analysis. In contrast, confocal microscopy enabled discrimination between the effects of sensitizers and irritants: sensitizer-treated samples showed internalized HLA-DR molecules aggregated in large vesicles with very bright fluorescence; irritant-treated samples were not different from untreated controls and showed compact HLA-DR molecules in small vesicles with diffuse fluorescence, and mostly localized in the submembranous zone. Electron microscopy demonstrated that sensitizer-treated LCs internalized HLA-DR molecules preferentially in lysosomes collected near the nucleus, whereas the irritant-treated and non-treated LCs internalized these molecules in the prelysosomes only near the cell membrane. We conclude that contact allergens and irritants induce distinct patterns of HLA-DR endocytosis, which may be useful for the development of in vitro screening tests.