After short term culture (2 to 3 days), Langerhans cells (LC) exhibit increased class II MHC Ag and become more potent APC than freshly obtained LC in primary allogeneic and syngeneic T cell activation. To determine whether in vivo LC undergo changes similar to cultured LC, we examined the phenotypic and functional characteristics of LC harvested from ear skin of naive mice painted with various haptens and primary irritants. At 24 h after application of 3% trinitrochlorobenzene, LC appear larger and exhibit more intense staining in epidermal sheets using anti-I-A antibodies, and there was a two- to threefold increase in I-A and I-E expression by LC using flow microfluorimetry analysis. CD45 Ag expression was not altered. Flow microfluorimetry profiles showed the presence of two different LC populations based on fluorescence intensity, i.e., one with the same Ia density as nontreated LC and the other (representing 22 to 50% of all LC) with a markedly enhanced Ia density, (i.e., a 10-fold increase in I-A and I-E). This phenotypic change was observed only with haptens, such as trinitrochlorobenzene, dinitrofluorobenzene, oxazolone, and cinnamic aldehyde. In contrast, application of 10 to 30% sodium lauryl sulfate or vehicle controls did not induce this change. Functionally, LC obtained from hapten-painted mice induced a two- to fivefold increase in 3[H]-TdR incorporation by syngeneic or allogeneic T cells, compared to equal numbers of LC from nontreated or vehicle-treated or sodium lauryl sulfate-treated mice. These phenotypic and functional changes that occur in vivo are therefore analogous to those seen when LC are cultured for short periods of time. Thus, activated LC appear in vivo in response to the epicutaneous application of haptens and may represent an essential step in hapten-specific sensitization.